International Journal of Molecular Sciences

Review Current Trends of Microfluidic Single-Cell Technologies

Pallavi Shinde 1,†, Loganathan Mohan 1,† , Amogh Kumar 1, Koyel Dey 1, Anjali Maddi 1, Alexander N. Patananan 2, Fan-Gang Tseng 3 , Hwan-You Chang 4, Moeto Nagai 5 and Tuhin Subhra Santra 1,* 1 Department of Engineering Design, Indian Institute of Technology Madras, Tamil Nadu 600036, India; [email protected] (P.S.); [email protected] (L.M.); [email protected] (A.K.); [email protected] (K.D.); [email protected] (A.M.) 2 Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA; [email protected] 3 Department of Engineering and System Science, National Tsing Hua University, Hsinchu City 30071, Taiwan; [email protected] 4 Department of Medical Science, National Tsing Hua University, Hsinchu City 30071, Taiwan; [email protected] 5 Department of Mechanical Engineering, Toyohashi University of Technology, Toyohashi 441-8580, Japan; [email protected] * Correspondence: [email protected] or [email protected] † These authors contributed equally to this work.



Received: 6 July 2018; Accepted: 27 September 2018; Published: 12 October 2018


Abstract: The investigation of human disease mechanisms is difficult due to the heterogeneity in gene expression and the physiological state of cells in a given population. In comparison to bulk cell measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. In this review, we describe the recent advances in single-cell technologies and their applications in single-cell manipulation, diagnosis, and therapeutics development.

Keywords: single-cell; lab-on-a-chip; microfluidics Bio-MEMS; manipulation; diagnosis; therapeutics; drug delivery

1. Introduction Cells are the fundamental operating units in living organisms. In multicellular organisms, cells communicate over small distances through direct contacts or over extended distances through chemical signals regulated by the microenvironment [1–3]. Such cell-cell and cell-matrix interactions are based on the gene expression of individual cells, with detrimental genetic aberrations leading to the development of diseases. The treatment of many diseases, such as diabetes, cancer, and neurodegenerative disorders, remains difficult due to the complex interaction of molecules and signaling pathways in and between cells [4–6]. To establish more effective treatments for these diseases, it is essential to target biomolecules and their respective cellular pathways to alter pathological conditions [7–9]. Over the past two decades, the ability to interrogate molecular and cellular pathways has improved rapidly [10–14] and has contributed to our better understanding of several diseases and the design of useful drugs [15–17]. However, as the throughput and complexity of various technologies designed to interrogate biological systems have increased, the cost and time associated

Int. J. Mol. Sci. 2018, 19, 3143; doi:10.3390/ijms19103143 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3143 2 of 47 with analyzing such large samples have also increased. Furthermore, many approaches to the study of cell biology involve the culturing of many cells together in a heterogeneous physiological state with different gene and biomolecule expression profiles. Data collected in such a bulk culture tend to lose low-frequency information [18,19] that may affect the accuracy of diagnosis and delay therapy [20–22]. Thus, it has become apparent that innovative methodologies are required to perform these analyses. The development of lab-on-a-chip, along with bio-micro electro mechanical systems (Bio-MEMS) or micro total analysis system (µTAS), may serve as a methodology that can provide high throughput data, with limited reagent consumption and assay complexity [23–26]. These systems are designed for single-cell assays at the 10–100 µm scale. Active monitoring of a single-cell in a well-controlled environment without the influences of nearby cells is essential to understand cell physiology. Single-cell technologies (SCT) are a set of novel techniques developed for studying single-cells and their interactions with other cells and the microenvironment in well-defined conditions [27–29]. In contrast to bulk analysis that provides average data for a population, SCT can identify different cellular characteristics and molecular dynamics in a cell population, which is particularly useful in characterizing tumor-initiating cells [28,29]. In order to analyze single-cell characteristics, data can be collected from individual cells in static or dynamic conditions so that phenotypic and functional alterations in diseased cells can be traced back to genetic aberration. Bioinformatics techniques can be used to handle the collection of such large amounts of diverse data generated from individual cells [27]. In the last two decades, the rapid development of Lab on a Chip has demonstrated that single cell analysis is a powerful technique for cell manipulation, diagnostics, and therapeutic developments [27,30,31]. Here, we demonstrate different physical approaches for single cell manipulation such as dielectrophoresis, optical, acoustic, and microfluidic microprobes. Furthermore, single cell separation and diagnosis techniques are discussed using microfluidic Bio-MEMS devices integrated with photomechanical, laser capture micro dissection, and nano-capillary chip technologies. Single cell therapeutic and analysis techniques are performed using electroporation [32], optoporation, mechanoporation [33], capillary electrophoresis coupled with laser-induced fluorescence detection, flow cytometry, mass spectrometry, high-resolution microscopy, the resonating microelectromechanical system (MEMS), electro neural nanowire device, and nanostraw system. Thus, this review article intends to provide an overview of currently available techniques for single cell manipulation, analysis, and their applications in diagnostic as well as therapeutic development. The research perspective, future prospects of SCT, and its applications will also be discussed.

2. Single-Cell Manipulation In-vitro cell growth conditions are often insufficient in reconstituting the cellular microenvironment at the high spatial resolution and cannot eliminate the interference of chemicals released from adjacent cells. Thus, establishing an ability to place a single-cell in the desired location through “single-cell manipulation” is essential for the investigation of cell physiology. A range of procedures for the physical displacement of single-cells has been developed. These procedures include fluidic-based [34–37], physical-based [38–41], electric field driven approaches such as dielectrophoresis (DEP), optoelectronic tweezers (OET) [42–48], and optical techniques such as optical tweezers [49–54]. The basic theories, advantages, and disadvantages of these technologies are briefly described below (Table1). Also discussed are recent developments in parallel single-cell manipulation. Int. J. Mol. Sci. 2018, 19, 3143 3 of 47

Table 1. Single-cell manipulation techniques.

Single-Cell S. No. Manipulation Features Advantages Disadvantages References Techniques Using Transportation of droplets Cell isolation, precise control is tedious. Droplet on small volumes, high Special care is required in [55–58] microfluidics. throughput screening. material choice for functions. Microfluidic DLD, Efficient cell separation based Sophisticated fabrication. [59–61] GEDI, GEM. on size, surface proteins. Hydrodynamic Same chip can be used for Tedious design [62–64] 1 Microfluidics pressure. multiple process. and fabrication. Fluidic microarray High throughput Less flexibility in single [65–68] chip. cell manipulation. cell manipulation. Cells can be displaced in the Small volume of cell Ink jet printing. [69] desired pattern. culture (8 µL) Increased throughput, useful Trapping and release rates Micropipette array. for transporting cells within are compromised to [70] different microenvironments. increase cell viability. Useful in handling inside Complex electrode 2 Electric field Dielectrophoresis. [71,72] microfluidic channels. fabrication. Contactless handling, Low throughput, Optical tweezers. label-free, high setup cost, [49–51] contamination-free. substrate-specifi.c 3 Optical energy Sophisticated instrument Increases throughput as setup, diffraction of light Multiple 3D compared to optical tweezers, limits the maximum [73] optical traps. cell detection is incorporated. number of cells that can be handled at one time. Label-free, contact less, 3D acoustic Sophisticated fabrication 4 Acoustic energy contamination free, [74] tweezer. and calibration process. safer as compared to the optical technique.

2.1. Fluidic-Based Manipulation

2.1.1. Droplet Microfluidics Droplet microfluidics involves compartmentalizing reactions by isolating molecules, particles or cells into droplets [55]. This is achieved by isolating the target particles in aqueous microdroplets, which are separated by immiscible oil or similar fluids. This two-phase system allows for a level of control on the picoliter scale. Droplet-based microfluidic devices are valuable tools for single-cell handling due to their capability of isolating cells for further analysis. These cells can be transported through microchannels for a variety of downstream applications. A lab on a chip for digitally controlling single cell encapsulation and independent aqueous reagent microdroplet reactors for screening cell viability and cytotoxicity has been proposed by Brouzes et al. [75]. Allowing higher flexibility and feasibility to handle microdroplets, the aqueous medium is replaced by hydrogels as discussed by Zhu et al. [76]. A comprehensive overview of droplet microfluidics has been discussed by Shang et al. [56]. Jenifer et al. [57] showed the application of droplet-based microfluidic platforms in high throughput screening of Human Embryonic Kidney cells (HEK293T) (Figure1). The microdroplets were generated by flow focusing a stream of cell suspension diluted in cell media, with perfluorinated oil. The concentration of nutrients inside the droplets was altered by varying the relative flow rates of the cell media and the cell suspension. The proliferation of both adherent and non-adherent cells in these microdroplets was observed for several days. Even after 14 days of incubation, greater than 90% of the drops remained intact, showing a very low degree of coalescence. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 46 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 46 in these microdroplets was observed for several days. Even after 14 days of incubation, greater than in these microdroplets was observed for several days. Even after 14 days of incubation, greater than Int.90% J. Mol.of the Sci. drops2018, 19 remained, 3143 intact, showing a very low degree of coalescence. 4 of 47 90% of the drops remained intact, showing a very low degree of coalescence.

Figure 1. Schematic diagram of the chip proposed by Jenifer et al. (A) The bounded rectangle shows Figure 1.1. SchematicSchematic diagramdiagram of of the the chip chip proposed proposed by by Jenifer Jenifer et al.et al. (A )(A The) The bounded bounded rectangle rectangle shows shows the the area where droplets containing cells are generated; (B) Mechanism of encapsulation of the cells in areathe area where where droplets droplets containing containing cells cells are generated;are generated; (B) Mechanism(B) Mechanism of encapsulation of encapsulation of the of cellsthe cells in the in the aqueous droplets. The red circles highlight the single cells in the droplets. Reprinted with aqueousthe aqueous droplets. droplets. The redThe circles red circles highlight highlight the single the cells single in the cells droplets. in the Reprinteddroplets. withReprinted permission with permission from [57]. frompermission [57]. from [57]. WhileWhile thethe aboveabove techniquestechniques introduce devices wherewhere isolated droplets are generated on-chip, While the above techniques introduce devices where isolated droplets are generated on-chip, YusofYusof etet al.al. reportedreported aa devicedevice forfor dispensingdispensing dropletsdroplets intointo otherother devicesdevices viavia anan inkjet-likeinkjet-like single-cellsingle-cell Yusof et al. reported a device for dispensing droplets into other devices via an inkjet-like single-cell printingprinting technologytechnology byby meansmeans ofof aa piezo-actuator [[69]69].. The schematic representation ofof inkjetinkjet printersprinters printing technology by means of a piezo-actuator [69]. The schematic representation of inkjet printers isis shownshown inin FigureFigure2 2[ [77].77]. While these devices are adept in terms of highly flexible flexible cell manipulation, is shown in Figure 2 [77]. While these devices are adept in terms of highly flexible cell manipulation, theythey areare limited limited in theirin their output output because because of the of use the of ause single of probe.a single Maintaining probe. Maintaining flexibility, accessibility, flexibility, they are limited in their output because of the use of a single probe. Maintaining flexibility, andaccessibility, increasing and the numberincreasing of probes the number favors higher of probes throughput favors in single-cellhigher throughput manipulation. in Thesingle-cell single accessibility, and increasing the number of probes favors higher throughput in single-cell probe-tipmanipulation. device The is appropriatesingle probe- fortip flexible device cellis appropriate manipulation. for flexible cell manipulation. manipulation. The single probe-tip device is appropriate for flexible cell manipulation.

FigureFigure 2.2. Schematic representation of Inkjet printers ejecting small droplets of cellscells andand hydrogelhydrogel Figure 2. Schematic representation of Inkjet printers ejecting small droplets of cells and hydrogel sequentiallysequentially toto buildbuild upup tissues.tissues. ReprintedReprinted withwith permissionpermission fromfrom [[77].77]. sequentially to build up tissues. Reprinted with permission from [77]. 2.1.2.2.1.2. MicrofluidicMicrofluidic DeterministicDeterministic LateralLateral DisplacementDisplacement TechnologyTechnology 2.1.2. Microfluidic Deterministic Lateral Displacement Technology UsingUsing microstructures to to control control the the fluidic fluidic field field is isa convenient a convenient way way to isolate to isolate single single cells. cells. The Using microstructures to control the fluidic field is a convenient way to isolate single cells. The Themajor major advantage advantage of using of using fluidic-based fluidic-based cell cell manipulation manipulation is isits its simplicity. simplicity. No No exogenous exogenous electric or magneticmajor advantage or optical of fieldsusing needfluidic-based to be applied, cell manipulation which can complicate is its simplicity. the instrument No exogenous design andelectric result or in unwanted effects on cells. Numerous microfluidic chips have used microfilters that prevent cells of Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 46 Int. J. Mol. Sci. 2018, 19, 3143 5 of 47 magnetic or optical fields need to be applied, which can complicate the instrument design and result in unwanted effects on cells. Numerous microfluidic chips have used microfilters that prevent cells certain sizes from passing through [78,79]. More complex microstructures such as micropillar arrays of certain sizes from passing through [78,79]. More complex microstructures such as micropillar can be used to separate cells of different sizes. One such design facilitated the isolation of circulating arrays can be used to separate cells of different sizes. One such design facilitated the isolation of tumor cells from blood samples [80]. The CTC-iChip developed by Karabacak et al. [59] provides a circulating tumor cells from blood samples [80]. The CTC-iChip developed by Karabacak et al. [59] novel way for the isolation of CTCs by using a two-stage magnetophoresis process (Figure3). In the provides a novel way for the isolation of CTCs by using a two-stage magnetophoresis process (Figure first stage, a continuous-flow system using deterministic lateral displacement (DLD) is used to separate 3). In the first stage, a continuous-flow system using deterministic lateral displacement (DLD) is used the nucleated cells from blood. This is done by specifying a critical deflection diameter (D ) for the to separate the nucleated cells from blood. This is done by specifying a critical deflection cdiameter DLD pillar arrays, such that the particles of interest have a diameter smaller than Dc and pass through (Dc) for the DLD pillar arrays, such that the particles of interest have a diameter smaller than Dc and the DLD pillar array without being deflected; while the particles having diameters larger than D pass through the DLD pillar array without being deflected; while the particles having diametersc would get deflected by the array. The second stage involves the use of inertial focusing to enable the larger than Dc would get deflected by the array. The second stage involves the use of inertial focusing precise lateral positioning of particles in a microfluidic channel, which then allows for highly efficient to enable the precise lateral positioning of particles in a microfluidic channel, which then allows for separation of target cells by magnetophoresis. highly efficient separation of target cells by magnetophoresis.

Figure 3.3.Schematic Schematic representation representation of CTCof CTC iChip iChip showing showing two stages: two stages: (1) iChip1 (1) usesiChip1 the LDLuses techniquethe LDL fortechnique separating for separating red blood red corpuscles blood corpuscles (RBCs), platelets(RBCs), platelets (PLTs) and (PLTs) free and beads free from beads the from whole the blood;whole (2)blood; iChip2 (2) iChip2 uses magnetophoresis uses magnetophoresis for separating for separati CTCng from CTC whitefrom white blood blood cells (WBCs). cells (WBCs). Reprinted Reprinted with permissionwith permission from [from59]. Copyright[59]. Copyright (2014) (2014) Springer Springer Nature. Nature.

Kirby et al. modifiedmodified DLD to develop a new microfluidicmicrofluidic devicedevice calledcalled geometricallygeometrically enhanced differential immunocapture (GEDI). (GEDI). This This device device ha hass a pillar a pillar array array designed designed to increase to increase targeted targeted cell cellto pillar to pillar collisions collisions during during the theflow flow path. path. This This is achieved is achieved by displacing by displacing the thepillar pillar rows rows with with an anincremental incremental offset. offset. These These pillars pillars are are fu functionalizednctionalized with with the the targeted targeted cell’s cell’s antigen recognizingrecognizing antibody. This This results results in in the the capture capture of oftargeted targeted cell cellss and and undesired undesired cell cellinteractions interactions are avoided. are avoided. This Thistechnique technique has hasbeen been successfully successfully used used for forprostrat prostrate,e, breast, breast, and and gastric gastric CTCs CTCs with with a 2–400-fold2–400-fold increaseincrease inin sensitivitysensitivity [[60].60]. Another antibody-based CTC cell trapping technique,technique, geometrical enhancedenhanced mixing (GEM), has been developed by Sheng et al.al. [[61]61] (Figure(Figure4 4).). ItIt consistsconsists ofof aa microfluidic microfluidic mixer mixer with with optimized optimized geometry forfor increasing increasing transverse transverse flow flow and and flow flow folding folding to increase to increase the cell the interaction cell interaction with antibody with functionalizedantibody functionalized surfaces. Thesurfaces. device The achieves device up achiev to 90%es cellup captureto 90% efficiencycell capture with efficiency 84% purity. with 84% purity. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 46 Int. J. Mol. Sci. 2018, 19, 3143 6 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 46

Figure 4. (a) Picture of a Microfluidic GEM chip; (b) Micrograph of the channel showing grooves FigureFigure 4. 4.(a )(a Picture) Picture of of a a Microfluidic Microfluidic GEMGEM chip;chip; (b) Micrograph of of the the channel channel showing showing grooves grooves insideinside the the channel channel useful useful in in cell cell capture; capture; capture; ( (cc)) Narrow NarrowNarrow groove (with(with dimensions) dimensions) showing showing captured captured cells cells (purple);(purple); ((dd ))(d CrossCross) Cross sectional sectional sectional view view view showing showing showing the thethe wide widewide groove groove (with (with(with dimensions). dimensions). dimensions). “Reproduced “Reproduced “Reproduced from from from [61 ] with[61][61] with permission with permission permission of The of of RoyalThe The Royal Royal Society Society Society of Chemistry”. ofof Chemistry”.Chemistry”.

2.1.3.2.1.3. Hydrodynamic Hydrodynamic Pressure Pressure Based Based Fluidic Fluidic ManipulationManipulation T ArakawaT Arakawa et et al. al. [[62]62 [62]] introduced introduced aa devicedevice thatthat captures cells cells in in small small trapping trapping micropockets, micropockets, enablingenabling further further single single cell cell processes processes suchsuch as as isolation, isolation, tagging,tagging, andand and single-cell single-cell single-cell rupture rupture rupture (Figure (Figure (Figure 5). 55). ). TheThe devicedevice device consistsconsists consists of of microchannelsof microchannels microchannels that that that are areare serpentine serpenserpentine in in shapein shapeshape and and and consist consist consist of of 64 of 64 or 64 or 512 or 512 trapping512 trapping trapping sites insites eachsites in channelineach each channel channel in series. in in series. The series. device The The allowsdevice device cell allowsallows trapping cell trapping followed followedfollowed by isolation, by by isolation, taggingisolation, andtagging tagging rupture and and in successionrupturerupture in ininsuccession thesuccession same channel in in the the the samesame cells channelchannel are captured thethe cells in. The are serpentine capturedcaptured channels’in.in. TheThe serpentine geometricserpentine channels’ parameters channels’ aregeometricgeometric optimized parameters parameters based on are cellare optimized sizeoptimized using based finitebased element onon cellcell si fluidicze using analysis. finitefinite element element Arakawa fluidic fluidic et al. analysis. observedanalysis. Arakawa Arakawa a capture rateet al.et of al.observed 70–83.2% observed a inacapture capture their experiments. rate rate of of 70–83.2% 70–83.2% The semicircular inin theitheir experiments. trapping The pocketsThe semicircular semicircular dimensions trapping trapping are decided pockets pockets as perdimensionsdimensions the size ofare are cells decided decided considered, as as per per tothe the prevent size size ofof the cellscells cells considered, from washing toto preventprevent away the duethe cells cells to thefrom from sudden washing washing flow away inaway the mainduedue to channel. tothe the sudden sudden Finally, flow flow cell in in rupturethe the main main is channel. channel. performed Finally,Finally, by injecting cell rupturerupture surfactant is is performed performed into the by mainby injecting injecting channel. surfactant surfactant The cell into the main channel. The cell membranes rupture and the cells’ contents are obtained by applying membranesinto the main rupture channel. and The the cell cells’ membranes contents are rupture obtained and bythe applying cells’ contents a negative are obtained pressure by through applying the a negative pressure through the collection channels. Thus, cell trapping, tagging and rupture can be collectiona negative channels.pressure through Thus, cell the trapping, collection tagging channels and. Thus, rupture cell can trapping, be performed tagging in and succession rupture can on the be performed in succession on the same device. sameperformed device. in succession on the same device.

Figure 5. Trapping of single cells using the device designed by Arakawa et al. The cells can either be Figurecaught 5. TrappingbyTrapping the drain of of single singlechannels cells cells or using usingcontinue the the deviceflowing device desi designedin gnedthe main by by Arakawa channel Arakawa aset etdenotedal. al. The The cells by cells the can canarrows. either either be becaught“Reproduced caught by bythe the drain from drain [62],channels channels with theor or continuepermission continue flowing of flowing AIP Publishing”.in in the the main main channel channel as as denoted denoted by by the the arrows. arrows. “Reproduced“Reproduced fromfrom [[62],62], withwith thethe permissionpermission ofof AIPAIP Publishing”.Publishing”. Int. J. Mol. Sci. 2018, 19, 3143 7 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 46

Bragheri and Osellame [63] [63] introduced a chip to enable single cell manipulation by varying pressures from two sources: an input sample cont containingaining the cell suspension, and a buffer sample (Figure 66).). TheThe ratioratio ofof thethe pressurespressures ofof inputinput flowingflowing fromfrom thesethese twotwo sourcessources isis thethe majormajor factorfactor determining the level of the buffer layer in thethe commoncommon channel.channel. Both the sources originate from different heights and since the flows flows are laminar, the levels of both the samples can be controlled by justjust altering their input input pressures. pressures. Lincoln et et al. [64] [64] showed that cell cellss or microbeads tend to flow flow at the bottom of a channel under low flow flow rates. Thus Thus,, the the positions positions of the cells in the common common channel can be manipulated using this device.

Figure 6. Schematic of different levels of the sample (distilled water) and the buffer (red food dye) in Figure 6. Schematic of different levels of the sample (distilled water) and the buffer (red food dye) in the common channel as per the ratio of the sample input pressure Ps and the buffer input pressure Pb the common channel as per the ratio of the sample input pressure Ps and the buffer input pressure Pb in the device designed by Bragheri and Osellame. (a) (Ps/Pb) = 1.85. (b) (Ps/Pb) = 1.6. (c) (Ps/Pb) = 1.2. in the device designed by Bragheri and Osellame. (a) (Ps/Pb) = 1.85. (b) (Ps/Pb) = 1.6. (c) (Ps/Pb) = 1.2. (d) (d) (Ps/Pb) = 1.85. Redrawn from [63]. (Ps/Pb) = 1.85. Redrawn from [63]. 2.1.4. Fluidic Microarray Chip for Cellular Manipulation 2.1.4. Fluidic Microarray Chip for Cellular Manipulation One of the problems faced when dealing with cells populations is that they typically are present in hugeOne numbers of the and problems are individually faced when vulnerable. dealing with Processing cells populations a large number is that of cellsthey renderstypically sophisticated are present inexperiments huge numbers lengthy and and are difficult individually to repeat vulnerable. when one cell Processing is handled a at large a time. number In addition, of cells one renders doesn’t sophisticatedwant to lose important experiments information lengthy and from difficult SCT during to repeat such procedures.when one cell Hence, is handled it becomes at a essentialtime. In addition,to manipulate one doesn’t the maximum want to lose possible important number info ofrmation cells simultaneouslyfrom SCT during while such beingprocedures. consistent Hence, in itmaintaining becomes essential their individuality. to manipulate Several the maximum researchers possible have number established of cells the simultaneously possibility of while developing being consistent2-D microwell in maintaining arrays on semiconductor their individuality. substrates Several or researchers micromachining have processesestablished to the achieve possibility a highly of developingparallel assay 2-D on microwell a microchip arrays [81–83 on]. semiconductor substrates or micromachining processes to achieveHolding a highly a cell parallel through assay a on glass a microchip pipette by [81–83]. suction flow is a standard technique for single-cell manipulationHolding a [ 84cell]. However,through a a glass recent pipette parallel by manipulation suction flow ofis single-cellsa standard bytechnique forming for an single-cell array was manipulationreported by Nagai [84]. etHowever, al. [70], who a recent described parallel a device manipulation that comprised of single-cells of 100 microprobesby forming an within array a chipwas reportedfor single-cell by Nagai manipulation. et al. [70], who Figure described7 shows a thedevice principle that comprised of this array-based of 100 microprobes platform, within where a each chip formicroprobe single-cell in manipulation. the array acted Figure as a micropipette. 7 shows the A pr suctioninciple force of this is created array-based by holding platform, the chip where over each the microprobedesired cell culturein the array matrix acted and as applying a micropipette. negative pressureA suction to force the chip is created through by a holding polydimethylsiloxane the chip over the(PDMS) desired pump. cell culture matrix and applying negative pressure to the chip through a polydimethylsiloxaneThe dimension of (PDMS) the probe pump. aperture and the suction pressure is optimized in such a way that individualThe dimension cells are of held the by probe each aperture probe. Aand microwell the suction array pressure is also is designed optimized such in such that a individual way that individualcells can be cells released are held in each by each well probe. on applying A microwell positive array pressure is also through designed the such PDMS that pump individual to the chip.cells canThis be study released may in promote each well a mechanical on applying model positive for minimallypressure through invasive the single-cell PDMS pump manipulation. to the chip. This study may promote a mechanical model for minimally invasive single-cell manipulation. Int. J. Mol. Sci. 2018, 19, 3143 8 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 46 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 46

FigureFigure 7. Schematic 7. Schematic representation representation of parallel of parallel manipulation manipulation of single-cells: of single-cells: The orange The orange arrows arrows represent represent the direction of movement of air on application of pressure and the red arrows represent theFigure direction 7. Schematic of movement representation of air on application of parallel of pressuremanipulation and theof redsingle-cells: arrows representThe orange the arrows direction the direction of the movement of cells due to applied pressure. (a) Pattern-based trapping of one type of therepresent movement the direction of cells dueof movement to applied of pressure. air on app (a)lication Pattern-based of pressure trapping and the of onered typearrows of represent single-cells; of single-cells; (b) Releasing the cells after transportation; (c) Suction of another type of single-cells; (b)the Releasing direction the of the cells movement after transportation; of cells due to (c applied) Suction pressure. of another (a) Pattern-based type of single-cells; trapping ( ofd) one Transport type (d) Transport of another type of cells for co-culture. Reprinted with permission from [70]. Copyright ofof another single-cells; type ( ofb) cellsReleasing forco-culture. the cells after Reprinted transportation; with ( permissionc) Suction of fromanother [70 type]. Copyright of single-cells; (2015) (2015) Springer Nature. Springer(d) Transport Nature. of another type of cells for co-culture. Reprinted with permission from [70]. Copyright (2015)Hong Springer et al. [36] Nature. and Chen et al. [37] effectively executed a cell-cell communication platform that canHong be used et al. to [ 36co-culture] and Chen a pair et al.of cells [37] in effectively one chamber. executed For effective a cell-cell single-cell communication pairing, Frimat platform et al. that Hong et al. [36] and Chen et al. [37] effectively executed a cell-cell communication platform that can[35] be used established to co-culture a new a paircellular of cells valving in one model chamber. to use For within effective a differential single-cell resistance pairing,Frimat microfluidic et al. [ 35] can be used to co-culture a pair of cells in one chamber. For effective single-cell pairing, Frimat et al. establishedcircuit. Microfluidic a new cellular control valving of cell model pairing to and use fusion within was a differentialstudied by Skelley resistance et al. microfluidic [34]. Cell fusion circuit. [35] established a new cellular valving model to use within a differential resistance microfluidic Microfluidicwas attained control in an array of cell format pairing once and the fusioncells were was paired studied [36,37]. by SkelleyThe flexibility et al. of [34 the]. design Cell fusion for the was attainedcircuit.use of in differentMicrofluidic an array cell format numberscontrol once of and cell the cell pairing cells types were andcan paired befusion improved [36was,37 studied]. using The flexibilitythese by Skelley chips. of et the al. design[34]. Cell for fusion the use of differentwas attainedDi Carlo cell in numbers et an al. array [65] and formatproposed cell once types a devicethe can cells be for were improved microf pairedluidic-based using [36,37]. these Theculture chips. flexibility of cells of in the ordered design arrays for the usewithDi of Carlo controlleddifferent et al. cell dynamic [65 numbers] proposed perfusion; and acell device shown types forincan Figure microfluidic-based be improved 8. They identifiedusing these culture important chips. of cells parameters in ordered for arraysthe Di Carlo et al. [65] proposed a device for microfluidic-based culture of cells in ordered arrays withdevice, controlled such as dynamic higher trapping perfusion; efficiency shown observed in Figure in8. case They of identified asymmetry important in offset distances parameters of rows for the with controlled dynamic perfusion; shown in Figure 8. They identified important parameters for the device,as compared such as higher to symmetrically trapping efficiency offset rows. observed They inalso case observed of asymmetry the ideal in depth offset distancesto vary with of rows the as device, such as higher trapping efficiency observed in case of asymmetry in offset distances of rows comparedaverage to size symmetrically of the cell. In their offset experiments rows. They with also HeLa observed cells, they the idealfound depth the single to vary cell initial with thetrapping average as compared to symmetrically offset rows. They also observed the ideal depth to vary with the sizerate of the to be cell. 70% In with their the experiments proposed device with and HeLa above cells, 85% they retention found theof the single cells cellin the initial trapped trapping sites. The rate to averagecells were size shown of the tocell. be In adherent their experiments to the PDMS with trappi HeLang cells, sites theyand onlyfound 5% the of single the cells cell were initial apoptotic trapping be 70% with the proposed device and above 85% retention of the cells in the trapped sites. The cells rateafter to 24 be hours, 70% with while the 1% proposed had undergone device and cell abovedivision. 85% retention of the cells in the trapped sites. The werecells shown were toshown be adherent to be adherent to the to PDMS the PDMS trapping trappi sitesng andsites onlyand only 5% of5% the of cellsthe cells were were apoptotic apoptotic after 24after h, while 24 hours, 1% had while undergone 1% had undergone cell division. cell division.

Figure 8. (A). Architecture of the system proposed by Di Carlo et al. The scale bar is 500 µm; (B). Diagram of the microfluidic chip and the trapping mechanism. “Reproduced from [65] with permission of The Royal Society of Chemistry”. FigureFigure 8. 8.(A ().A). Architecture Architecture of of the the system system proposedproposed by Di Di Carlo Carlo et et al. al. The The scale scale bar bar is is500 500 µm;µm; (B). (B ). DiagramDiagram of theof microfluidicthe microfluidic chip andchip theand trapping the tra mechanism.pping mechanism. “Reproduced “Reproduced from [65 from] with [65] permission with Wlodkowic et al. [66] introduced a microfluidic platform for screening of anticancer drugs of Thepermission Royal Societyof The Royal of Chemistry”. Society of Chemistry”. against single cells trapped in arrays. The device consists of an array of traps fabricated with PDMS (Figure 9). It showed a trapping efficiency of 10–20% for both U937 and HL60 cells in their WlodkowicWlodkowic et al.et [al.66 ][66] introduced introduced a microfluidic a microfluidic platform platform forscreening for screening of anticancer of anticancer drugs drugs against singleagainst cells single trapped cells in trapped arrays. in The arrays. device The consists device of consists an array of ofan trapsarray fabricatedof traps fabricated with PDMS with (Figure PDMS9 ). (Figure 9). It showed a trapping efficiency of 10–20% for both U937 and HL60 cells in their Int. J. Mol. Sci. 2018, 19, 3143 9 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 46 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 46 experimentsIt showed a trappingand over efficiency 85% of of the 10–20% cells forinitially both U937trapped and HL60retained cells their in their position experiments even after and overcell experiments85% of the cells and initially over 85% trapped of the retained cells initially their position trapped even retained after cell their perfusion. position Wlodkowic even after etcell al. perfusion. Wlodkowic et al. also discussed the potential of the device in proliferation of the cells after trappingalso discussed [67]. Thus, thepotential the microfluidic of thedevice device inallows proliferation for real-time of the trapping cells after andtrapping screening [67 of]. cells Thus, in trappingthe microfluidic [67]. Thus, device the allows microfluidic for real-time device trapping allows for and real-time screening trapping of cells inand small screening populations. of cells in small populations.

Figure 9. CAD schematic representation of the microfluidic device proposed by Wlodkowic et al. Figure 9. CADCAD schematic schematic representation representation of of the the microflu microfluidicidic device device proposed proposed by by Wlodkowic Wlodkowic et et al. al. showing the traps in the triangular array. Reprinted with permission from [66]. Copyright (2009) showing the traps in the triangulartriangular array.array. ReprintedReprinted with permission from [[66].66]. Copyright Copyright (2009) (2009) American Chemical Society. American Chemical Society.

Xu et al. [68] [68] proposed a novel cell trapping mi microfluidics-basedcrofluidics-based device and developed a robust framework to to identify and optimize parameters to to maximize trapping efficiency efficiency in the proposed device (Figure 10).10). The The device device first first captures captures microsph microsphereseres flown flown into it via the the PDMS-fabricated PDMS-fabricated traps followed by capture of target cells on thethe trappedtrapped microspheres. The microspheres have embedded receptors to capture the cells [85]. [85]. The optimized dev deviceice reported reported a a trapping trapping efficiency efficiency of 99% and a high trap density of 1438 traps/mm22 inin their their experiments. experiments. high trap density of 1438 traps/mm2 in their experiments.

Figure 10. Schematic representation of the microfluidic microsphere-trap array. (a) Layout (top view): Figure 10. SchematicSchematic representation representation of of the the micr microfluidicofluidic microsphere-trap microsphere-trap array. array. ( a)) Layout Layout (top (top view): view): Microfluidic channels with hydrodynamic trap arrays. The channels are connected by a common inlet MicrofluidicMicrofluidic channels with hydrodynamic trap arrays.arrays. The channels are connected by a common inlet and a common outlet. Liquid solution carrying the microspheres flows from the inlet and through the and a common outlet. Liquid Liquid soluti solutionon carrying carrying the microspheres microspheres flow flowss from the inlet and through the chamber; (b) Trapping mechanism. Reproduced from [68] with the permission of AIP Publishing. chamber; ( b) Trapping mechanism.mechanism. Reproduced fromfrom [[68]68] withwith thethe permissionpermission ofof AIPAIP Publishing.Publishing.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 46

Int.2.2. J. Electric Mol. Sci. Field-Driven2018, 19, 3143 Manipulation 10 of 47 Manipulation and patterning of cells can be achieved through the application of dielectrophoretic (DEP) conditions using non-uniform electric fields [71,72]. Hunt et al. 2006 & 2.2. Electric Field-Driven Manipulation Kodama et al. 2013 reported on dielectrophoretic tweezer techniques as an alternative to suction for cell manipulationManipulation [86,87]. and patterning Lee et al. of utilized cells can the be DEP achieved tweezer through technolo thegy application for studying of dielectrophoretic particle surface (DEP)interaction conditions in microfluidic using non-uniform devices, which electric further fields developed [71,72]. Huntinto a etforce al. spectroscopy 2006 & Kodama technique et al. 2013[88]. reportedKim et al. on automated dielectrophoretic the DEP tweezer tweezer-based techniques force as an spec alternativetroscopy to technique suction for to cell manipulate manipulation particles[86,87 in]. Lee3D [89]. et al. utilized the DEP tweezer technology for studying particle surface interaction in microfluidic devices, which further developed into a force spectroscopy technique [88]. Kim et al. automated the DEP2.3. Optical tweezer-based Manipulation force spectroscopy technique to manipulate particles in 3D [89].

2.3. OpticalOptical Manipulation tweezers are widely used for single cell optical manipulation. Figure 11 shows the schematic diagram of optical tweezers. When light strikes an object, its scattering is always accompaniedOptical tweezers with some are widelymomentum used transfer for single to cellthe opticalobject manipulation.as per the law Figure of conservation 11 shows theof schematicmomentum. diagram This implies of optical that while tweezers. some photons When lightin light strikes beams an getobject, scattered its off scattering the incident is alwayssurface accompaniedof the object, withsome some impart momentum momentum transfer to it toin thethe objectdirection as per of thethe lawbeam. of conservationWhen a dielectric of momentum. object is Thisirradiated implies by that a Gaussian while some laser photons beam, dielectric in light beams particles get scatteredare attracted off the towards incident the surface center of the object,beam, somesince it impart is the momentumregion where to the it in electric the direction field is st ofrongest. the beam. Thus, When any a small dielectric displacement object is irradiatedaway from by the a Gaussiancenter of laserthe beam beam, causes dielectric a restoring particles force are attractedgenerated towards by the theelectric center field of the gradient beam, sinceto act iton is thethe regionparticle, where pushing the electricit back field towards is strongest. the center Thus, of anythe smallbeam. displacement In addition, awaythe net from pressure the center from of the beamphotons causes in the a restoring direction force of the generated beam applies by the a electric positive field force gradient on the to particle act on thealong particle, the direction pushing of it backbeam towards propagation the center [90,91]. of the beam. In addition, the net pressure from the photons in the direction of the beamThis appliestechnique a positive can be forceeffectively on the employed particle along in pr theecise direction single cell of beam handli propagationng. The advantages [90,91]. of usingThis optical technique methods can have be effectively been reported employed by Chiou in precise et al. single 2005, cell Mirsaidov handling. etThe al. 2008, advantages and Juan of using et al. optical2011. However, methods optical have been tweezers reported are byprone Chiou to etlight al. 2005,diffraction Mirsaidov and impose et al. 2008, limitations and Juan on et available al. 2011. However,substratesoptical when handling tweezers aremultiple prone cells to light at the diffraction same time and [49–51]. impose Liberale limitations et al. on [92] available presented substrates a new whenapproach handling to integrating multiple cells optical at the trapping same time with [49 mi–51crofluidic]. Liberale devices et al. [92 for] presented on-chip manipulation a new approach and to integratinganalysis by optical using trapping miniaturized with microfluidic optical tweezers. devices forTheir on-chip device manipulation shows great and analysispotential by in using cell miniaturizedmanipulation optical and screening tweezers. due Their to deviceits versatility shows greatand ease potential of fabrication, in cell manipulation whilst also and ensuring screening cell duesafety. to its versatility and ease of fabrication, whilst also ensuring cell safety.

Figure 11.11. Schematic diagram of opticaloptical tweezerstweezers usedused toto manipulatemanipulate singlesingle cells.cells. ReprintedReprinted withwith permissionpermission fromfrom [[91].91]. CopyrightCopyright (2016)(2016) SpringerSpringer Nature.Nature. Int. J. Mol. Sci. 2018, 19, 3143 11 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 11 of 46

ToTo achieveachieve parallelparallel manipulationmanipulation ofof cells,cells, WangWang etet al.al. [[73]73] combinedcombined hydrodynamicshydrodynamics withwith thethe holographicholographic opticaloptical tweezingtweezing operationoperation to createcreate multiplemultiple 3D3D opticaloptical trapstraps toto separateseparate cellscells fromfrom aa mixturemixture (Figure(Figure 1212).). TheThe 3D3D opticaloptical trapstraps areare integratedintegrated intointo aa microfluidicmicrofluidic devicedevice withwith anan arrayarray ofof microwellsmicrowells toto dockdock cellscells thatthat passpass overover it through a microfluidicmicrofluidic channel placed on a motorized stage. TheThe dockeddocked cellscells areare thenthen observedobserved throughthrough aa microscopemicroscope thatthat scansscans the arrayarray to capture the image. UsingUsing anan imageimage processingprocessing technique,technique, thethe desireddesired cellscells areare detecteddetected andand anan opticaloptical traptrap isis createdcreated toto traptrap specificspecific cells using using a a continuous continuous wave wave laser laser source. source. Multiple Multiple traps traps are are created created depending depending on the on thenumber number of cells of cells detected. detected. The Z The stageZ stageof the ofobjective the objective moves moves vertically vertically upwards upwards to levitate to levitatethe trapped the trappedcells. The cells. motorized The motorized stage then stage moves then in moves an X–Y in direction an X–Y direction to transport to transport the cells to the the cells desired to the location. desired location.The entire The process entire is process controlled is controlled by software. by software. Wang et Wang al. reported et al. reported a successful a successful levitation levitation rate of rate78.5 of± 5.4% 78.5 ±and5.4% a successful and a successful transfer transfer rate of 97 rate ± 1.41% of 97 ± using1.41% this using proposed this proposed system [93]. system [93].

FigureFigure 12.12. SchematicSchematic representationrepresentation ofof cellcell manipulation using computer-controlled motorized stage. RedrawnRedrawn fromfrom [[93].93].

ThisThis techniquetechnique achievesachieves thethe parallelparallel manipulationmanipulation ofof cells.cells. However,However, itit requiresrequires aa sophisticatedsophisticated experimentalexperimental setup.setup. In addition,addition, the numbernumber ofof opticaloptical trapstraps thatthat cancan bebe generatedgenerated isis limitedlimited byby thethe maximummaximum laserlaser power.power. WangWang etet al.al. [[94]94] introducedintroduced aa systemsystem integratingintegrating opticaloptical tweezerstweezers intointo microfluidicmicrofluidic technologytechnology forfor cellcell isolation,isolation, transporttransport andand depositiondeposition inin aa non-invasivenon-invasive mannermanner (Figure(Figure 1313).). TheirTheir systemsystem usesuses digitaldigital imageimage processingprocessing toto identifyidentify importantimportant featuresfeatures suchsuch asas cellcell sizesize andand fluorescencefluorescence toto identifyidentify targettarget cells.cells. TheThe opticaloptical trapstraps cancan bebe generatedgenerated byby theirtheir systemsystem atat anyany positionposition insideinside thethe regionregion ofof interestinterest toto traptrap thethe cellscells onceonce theythey areare detecteddetected byby thethe imageimage processingprocessing module.module. To capturecapture thethe cells,cells, thethe fluidfluid dragsdrags force,force, andand thethe opticaloptical trappingtrapping forceforce mustmust neutralizeneutralize eacheach otherother soso thatthat thethe cellcell movesmoves atat aa constantconstant velocityvelocity andand cancan bebe movedmoved fromfrom thethe samplesample flowflow toto thethe bufferbuffer flowflow usingusing thethe opticaloptical tweezerstweezers module.module. TheyThey demonstrateddemonstrated thethe workingworking ofof thisthis systemsystem usingusing HumanHuman EmbryonicEmbryonic StemStem cellscells andand reportedreported highhigh puritypurity andand recoveryrecovery raterate ofof thethe targettarget cells cells from from the the input input sample. sample. Int. J. Mol. Sci. 2018, 19, 3143 12 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 46 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 46

Figure 13.13. SchematicSchematicSchematic representationrepresentation ofof the the cellcell sortingsortsortinging procedure.procedure.procedure. ReproducedReproduced Reproduced fromfrom [94][94] [94] withwith permission of The RoyalRoyal SocietySociety ofof Chemistry.Chemistry.

2.4. Acoustic Based Mainpulation 2.4. Acoustic Based Mainpulation Ding et al. introduced the first first acoustic tweezer tweezerss (Figure 14),14), which showed precision close to thosethose of of opticaloptical tweezerstweezers whilewhile havinghaving a a powerpower densitydensity ordersorders ofof magnitudemagnitude lesser than than thosethose ofof optical tweezers (10,000,000 times lesser) and optoel optoelectronicectronic tweezers (100 times lesser), thus making acoustic tweezers way more biocompatible.biocompatible. The de devicevice was employed in 2D2D acousticacoustic manipulationmanipulation of HeLa HeLa cells cells and and micro-organisms micro-organisms by by real-time real-time control control of a of standing a standing surface surface acoustic acoustic wave wave field. field. The Thedevice device showed showed the ability the ability of moving of moving cells across cells the across platform the platform at a very athigh a very speed high of up speed to 1600 of µm/s. up to µ 1600They m/s.used Theypolystyrene used polystyrene microparticles microparticles to show to how showhow the howthe devicedevice the device enabledenabled enabled preciseprecise precise andand and intricateintricate manipulation on the 2D platform [[95].95].

Figure 14. Schematic diagram showing the mechanism of the device proposed by Ding et al. Figure 14.14.Schematic Schematic diagram diagram showing showing the mechanismthe mechanism of the of device the proposeddevice proposed by Ding etby al. Ding Permission et al. Permission to reprint obtained from PNAS [95]. toPermission reprint obtained to reprint from obtained PNAS from [95]. PNAS [95].

Another technique to manipulate multiple cellscells waswas demonstrated byby GuoGuo et al. They developed 3D acoustic tweezers to manipulate microparticles and cells (Figure 15).15). The figure figure shows electrodes used to create surface acoustic waves and the regi regionon of operation. The device creates standing waves by superimposing surface acoustic waves to form 3D 3D trapping trapping nodes. nodes. To To achieve achieve in-plane in-plane movement, movement, theythey controlled controlled the the phasephase shift shift of of thethe standingstanding wavewawave and the amplitude of the wave controlled the orthogonal movements [[74].74]. Int. J. Mol. Sci. 2018, 19, 3143 13 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 13 of 46

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 13 of 46

FigureFigure 15. 15Schematic. Schematic representation representation of of 3D 3D acousticacoustic tweezerstweezers showing showing particle particle trapping. trapping. The The solid solid arrows represent the movement of cell in X, Y and Z direction. The dotted arrows show an enlarged arrowsFigure represent 15. Schematic the movement representation of cell of in 3D X, Yacoustic and Z direction.tweezers showing The dotted particle arrows trapping. show The an enlarged solid view of cell location on chip. Permission to reprint obtained from PNAS [74]. viewarrows of cell represent location the on chip.movement Permission of cell in to X, reprint Y and obtainedZ direction. from The PNAS dotted [74 arrows]. show an enlarged view of cell location on chip. Permission to reprint obtained from PNAS [74]. 3. Single-Cell3. Single-Cell Technologies Technologies (SCT) (SCT) for for Research Research andand DiagnosisDiagnosis 3.In Single-Cell orderIn order to treatto Technologies treat diseases diseases properly, (SCT) properly, for we Researchwe need need toto and understandunderstand Diagnosis the the genetic genetic information information and and metabolic metabolic pathwayspathwaysIn oforder abnormalof abnormalto treat diseases cells. cells. Efficient Efficientproperly, and and we sensitiveneedsensitiv to understande detection theof of the thegenetic chemical chemical information components components and metabolic within within a a single-cellpathwayssingle-cell is still ofis stillabnormal challenging. challenging. cells. InEfficient In this this section, section, and sensitiv wewe discussdiscusse detection some some ofof of thethe the chemicalrecently recently developedcomponents developed devices within devices for a for detecting abnormal cells from a bulk of cells (Table 2). detectingsingle-cell abnormal is still challenging. cells from a In bulk this of section, cells (Table we discuss2). some of the recently developed devices for detecting abnormal cells from a bulk of cells (Table 2). 3.1. Single-Cell Separation and Detection 3.1. Single-Cell Separation and Detection 3.1. Single-CellFlow cytometry Separation is andan Detectionexceptional tool for single-cell sorting based on cell properties or Flow cytometry is an exceptional tool for single-cell sorting based on cell properties or fluorescence fluorescenceFlow cytometry markers (1974)is an [96].exceptional Fluorescence-Assis tool for single-cellted Cell Sortingsorting (FACS)based ison a celltype properties of cytometry or markerswhere (1974)fluorescent-labelled [96]. Fluorescence-Assisted cells can be separated Cell from Sorting a heterogenous (FACS) is mixture a type of of cells, cytometry on a cell-by- where fluorescent-labelledfluorescence markers cells (1974) can be [96]. separated Fluorescence-Assis from a heterogenousted Cell Sorting mixture (FACS) of cells, is a ontype acell-by-cell of cytometry basis wherecell basis fluorescent-labelled (Figure 16). One cellsof the can major be separated advantages from of a heterogenousFACS is its potential mixture toof measurecells, on amultiple cell-by- (Figure 16). One of the major advantages of FACS is its potential to measure multiple characteristics cellcharacteristics basis (Figure of cells 16). basedOne ofon thethe fluorescencemajor advantages specificities of FACS of the is targetits potential constituents to measure of the cellsmultiple [97]. of cells based on the fluorescence specificities of the target constituents of the cells [97]. FACS also characteristicsFACS also provides of cells single based cell on datathe fluorescence at extremel yspecificities high throughput. of the target However, constituents DNA content of the cells analysis [97]. provides single cell data at extremely high throughput. However, DNA content analysis using FACS FACSusing FACSalso provides has shown single variability cell data in atprognostic extremel yresults, high throughput. hence the technique However, is notDNA very content well acceptedanalysis hasusingfor shown DNA FACS variability analysis has shown of insingle prognosticvariability cells [98]. in results, prognostic hence results, the techniquehence the technique is not very is not well very accepted well accepted for DNA analysisfor DNA of single analysis cells of [single98]. cells [98].

Figure 16. Basic Working Principle of a Flow Cytometer. “Reproduced from [99] with the permission of Taylor & Francis”. FigureFigure 16. 16.Basic Basic Working Working Principle Principle of of a a Flow Flow Cytometer.Cytometer. “Reproduced “Reproduced from from [99] [99 with] with the the permission permission of Taylorof Taylor & Francis”. & Francis”. Int. J. Mol. Sci. 2018, 19, 3143 14 of 47

Table 2. Single-cell diagnosis techniques.

Single-Cell S. No. Diagnosis Features Advantages Disadvantages References Techniques Using High throughput Not reliable for Flow cytometry, technique to analyze intracellular diagnosis. A microfluidic technique to heterogeneous cell Spectral overlap sort cells based on their size, [100] population. observed in screening granularity, Gives high accuracy of multiple and fluorescence (FACS). in sorting. parameters. Cell affinity micro Targeted cells sorted Low throughput. chromatography, based on surface Limited by Surface a technique based on proteins. interaction. [101] 1 Microfluidics binding with surface Higher sensitivity Complex cell retrieval immobilized ligands. and selectivity. mechanism. Microreactors for individual cells within a Huge data analysis Droplet microfluidics. [102] droplet, rare cell required. detection possible. Rare cell isolation is possible. Micro vortex. Purity of output is low. [103] High throughput is possible. Limited to cells Dielectrophoresis technique Useful in the study of showing a response to 2 Electric field uses non-uniform neural cells, cancer cells. [104] electric fields. electric field. High efficiency. Low throughput. single-cell captured per microfluidic chamber allows tracking of individual cells. Photomechanical cell Surface proteins are Low throughput. [105] detachment. preserved. Expensive setup. Genetic analysis can provide information 3 Optical energy about key altering genes in cell lineage. Allows live single-cell isolation. Requires good optical Laser capture Even cell organelles can resolution. [106] microdissection. be isolated. Expensive setup. Contactless technique. High efficiency. Based on surface MACS uses functionalized markers allows high Low throughput. 5 Magnetic energy super-paramagnetic selectivity and Cell retrieval is [107] nanoparticles to tag cells. sensitivity. difficult. Allows sorting of multiple target cells. In Digital droplet, PCR sample is fractionated into droplets using water oil Very high accuracy and emulsion droplet technology. sensitivity in detection Lengthy process. Each droplet is then allowed of rare DNA target. Redesigning of the 6 Omics technology [108] to react with the specific Quantification of the process required if primer and probe. concentration of targeted results are inconsistent. The droplets showing cell is possible. positive reaction are quantified.

Hence, droplet microfluidics for single-cell isolation fiber optics for target cell detection and lab-on-chip devices are combined to develop multiple cell sorting and collection through multiple channels with higher throughput [109]. Yet, for downstream single-cell analysis, the technique to separate targeted single-cells from bulk is essential. Separating cells and collecting them in 96 or 384 plate microwells is possible with FACS. However, cell sorting microfluidic devices based on antigen-antibody interactions have very high target specificity. Int. J. Mol. Sci. 2018, 19, 3143 15 of 47

Fluorescent activated cell sorters and magnetically activated cell sorters to detect and separate cells are available [110,111]. However, they cannot be used directly for substrate-attached cells without disrupting their microenvironment. In addition, the lineage of cells is lost in these techniques. However, micro array-based chip technology can provide both high throughput and preservation of cell lineage. Cell detachment from a substrate in routine cell culture is generally achieved by the use of trypsin. However, extracellular surface proteins are lost in the process. A physical detachment of cells from the substrate can circumvent the loss of surface proteins. This can be useful in the downstream study of cell surface properties. Keeping a track of cell lineage can be very useful in the study of cell differentiation and cell modification after division in diseased cells by acquiring comparative data from sister cells. Moreover, the influence of specific factors on sister cell development can be studied [112] and valuable data on the origin of genetic aberrations can be garnered [113]. However, such cell lineage tracking using conventional techniques is laborious and monotonous. Improving on traditional dish-based approaches, the latest progress in microfluidics facilitates the monitoring and isolation of thousands of single-cells on a chip with high resolution [114–118]. Such approaches have allowed for the microscopical observation of single-cell self-renewal and differentiation processes [119,120]. While single-cell monitoring offers valuable information, microfluidic methods are developing numerous techniques to recover healthy individual cells for genotypic investigations, necessary for reducing complex cell rejuvenation and differentiation pathways [121]. In cell detachment procedures like poly (N-isopropylacrylamide) (PNIPAAm)-based detachment or trypsinization, all cells are isolated from the whole substrate without any specific cell isolation [122]. In photodegradation, photo-acid-generating cell culture substrates have lower specificity and allow improved cell release. However, this approach creates acidic by-products with potentially toxic effects and possibly disturbs cell expression and behavior [123]. Other approaches using localized trypsin or capillary vacuums have been demonstrated; though these are still restricted to open substrates [124,125]. Optical methods using infrared laser irradiation resulted in very poor cell viability owing to heat-induced cell necrosis [126]. Recently, single-cell detachment through laser-generated focused ultrasound (LGFU) was also reported [127,128]. The focused ultrasound-induced single bubble cavitational disruption (<100 µm precision) to separate target cells. These methods are also restricted to open substrates. To overcome these drawbacks, Chen et al. developed a novel device [105,128] through which it is possible to preserve cell lineage data. Moreover, individual sister cells can be detached from the substrate without disturbing the microenvironment and can be collected for downstream analysis. Using this approach, gene expression signatures from sister cells were successfully compared and distinguishing factors were identified. The entire process was honed to consume less time and labor. Figure 17 shows the schematic view of a single-cell detachment set-up. The composite material was composed of carbon nanotubes (CNT) and PDMS. Cells were cultured on the composite film. A targeted cell was then irradiated with a single laser pulse for 7 ns and a spot diameter of 10 µm. In response to the optical energy, the CNTs act as a transducer to convert the photo irradiation into thermal energy and finally produce microbubbles. These microbubbles ruptured the PDMS film to exert a shear stress of 600 Pa on the cells seeded on top of the composite material, sufficient to induce single-cell detachment. The further expansion of the microbubble caused lateral displacement of the targeted cell. The laser beam was focused over a region of 10 µm in diameter, conducive to single-cell detachment. Furthermore, this approach can be honed to generate local detachment of a cell section. Upon integrating the composite film with the microchannel array, the detached cells were successfully retrieved. To study the sister cells, a microchamber was fabricated with microchannels for cell retrieval. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 16 of 46

Upon integrating the composite film with the microchannel array, the detached cells were successfullyInt. J. Mol. Sci. 2018 retrieved., 19, 3143 To study the sister cells, a microchamber was fabricated with microchannels16 of 47 for cell retrieval.

Figure 17. SchematicSchematic diagram diagram of of a single-cell detachment setup. ( (aa)) Cells Cells were were cultured cultured in in the micromicrofluidicfluidic chamber coated with the CNTCNT−PDMSPDMS composite. composite. A A short short pulse pulse laser laser is is used to detach the target cell; (b) MicrofluidicMicrofluidic chamberchamber and channelchannel forfor cellcell capturecapture andand retrieval.retrieval. Reprinted with permission from [[105].105]. Copyright (2017)(2017) American Chemical Society.

3.2. Antibody-Based Single-Cell Screening Flow cytometry is capable of detecting and sortingsorting cells, based on theirtheir size, shape, surface proteins and and fluorescence fluorescence markers. markers. Over Over the the past past three three decades decades [129] [129, many], many methods methods have havebeen developedbeen developed to detect to detect circulating circulating fetal fetal nucleated nucleated cells cells (CFNC); (CFNC); for for example, example, high-throughput microscopy [130,131],[130,131], filtrationfiltration [132[132],], magnetic-activatedmagnetic-activated cellcell sortingsorting (MACS)(MACS) [[133,134],133,134], gradient centrifuge [135,136], [135,136 ],fluorescence-activated fluorescence-activated cell sort celling sorting (FACS) [137,138], (FACS) [and137 ,microchip138], and technologies microchip [139,140].technologies These [139 techniques,140]. These can techniques isolate individual can isolate cells individual for further cells analysis. for further analysis. Liquid biopsy shows significant significant promise for future diagnosis. However, detection and isolation of targeted individual cells from millionsmillions of cells remains a challenge. The detection and capture of a targeted circulatingcirculating cellcell isis a a step step of of critical critical significance significance after after obtaining obtaining a liquida liquid biopsy biopsy sample. sample. Such Such an anapproach approach prevents prevents the needthe need for invasive for invasive biopsies biopsi thates are that frequently are frequently required required for prognosis, for prognosis, therapy therapyassessment, assessment, and proteomic and proteomic and genetic and studies.genetic studies. However, However, this method this method has drawbacks has drawbacks that include that poorinclude detection poor detection and separation and separation with low cellwith density low cell (one density cell per (one billion cell blood per billion cells) and blood heterogeneity. cells) and heterogeneity. 3.2.1. NanoVelcro Microchip 3.2.1.In NanoVelcro contrast to Microchip current rare-cell sorting methodologies, “Nano Velcro” rare-cell assays may significantlyIn contrast improve to current the performance rare-cell sorting of rare-cell meth enrichmentodologies, from“Nano blood Velcro” [141, 142rare-cell]. By incorporating assays may significantlythe Nano Velcro improve substrate the performance with a superimposed of rare-cell microfluidic enrichment chaotic from blood mixer, [141,142]. a Nano By Velcro incorporating microchip thecan Nano be produced. Velcro substrate The microchip with a has superimposed been validated microfluidic with high chaotic specificity mixer, and a sensitivity Nano Velcro for computingmicrochip canCirculating be produced. Tumor CellsThe microchip (CTCs) in differenthas been solid validat tumors,ed with including high CTCsspecificity of lung and cancer sensitivity [143–145 for], computingkidney cancer Circulating [146], pancreatic Tumor Cells cancer (CTCs) [147], in and different prostate solid cancer tumors, [148– including150]. CTCs CTCs have of been lung isolated cancer [143–145],by Nano Velcro kidney microchips cancer [146], coated pancreatic with electrospun cancer [147], poly and (lactic- prostateco-glycolic cancer acid) [148–150]. (PLGA) CTCs nanofibers, have coupled with a commercial laser capture microdissection (LCM) technique. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 17 of 46 Int. J. Mol. Sci. 2018, 19, 3143 17 of 47 been isolated by Nano Velcro microchips coated with electrospun poly (lactic-co-glycolic acid) (PLGA) nanofibers, coupled with a commercial laser capture microdissection (LCM) technique. The non-invasive technique for prenatal diagnosis developed by Hou et al. [151] involved the The non-invasive technique for prenatal diagnosis developed by Hou et al. [151] involved the fabrication of a microchip that acted as a Nano Velcro to detect circulating fetal nucleated cells fabrication of a microchip that acted as a Nano Velcro to detect circulating fetal nucleated cells (CFNC). Figure 18 schematically illustrates the structure of the Nano Velcro microchip, which is, (CFNC). Figure 18 schematically illustrates the structure of the Nano Velcro microchip, which is, an an array of nanopillars designed with optimum pillar dimensions. Anti–Ep Cam antibody was used array of nanopillars designed with optimum pillar dimensions. Anti–Ep Cam antibody was used to to functionalize the substrate to capture CFNC in the maternal blood. After the maternal blood was functionalize the substrate to capture CFNC in the maternal blood. After the maternal blood was gradient centrifuged for red blood cell depletion, it was passed through a chaotic mixer. The Nano gradient centrifuged for red blood cell depletion, it was passed through a chaotic mixer. The Nano Velcro and the chaotic mixer easily slide inside a metal sandwich frame, which is customized to align Velcro and the chaotic mixer easily slide inside a metal sandwich frame, which is customized to align and hold two components together. Upon passing the sample through the chaotic mixer, within and hold two components together. Upon passing the sample through the chaotic mixer, within the initialthe initial three three lanes lanes itself, itself, the CFNCs the CFNCs were werecaptured captured in the in Nano the Nano Velcro. Velcro. The LCM The LCMtechnique technique was used was toused focus to on focus each on individual each individual CFNC CFNCto separate to separate each single-cell each single-cell from the from Nano the Velcro Nano chip Velcro for chipgenetic for testing.genetic testing.

Figure 18. Anti-EpCAM functionalized nano Velcro microchip is assembled using a chip holder to Figurehold together 18. Anti-EpCAM a chaotic mixer functionalized on top of annano imprinted Velcro microchip PLGA Nano is assembled Velcro substrate. using a “Reprinted chip holder with to holdpermission together from a chaotic [151]. Copyrightmixer on top (2017) of an American imprinted Chemical PLGA Nano Society.” Velcro substrate. “Reprinted with permission from [151]. Copyright (2017) American Chemical Society.” 3.2.2. Microarray Chip Based Screening 3.2.2. Microarray Chip Based Screening Screening is the task of separating cells into numerous streamlines based on the distinction of theirScreening physical oris chemicalthe task of properties separating and cells obtain into them numerous through streamlines specific outlets. based This on the phenomenon distinction can of theirbe naturally physical observed or chemical in blood proper capillariesties and obtain wherein them red through blood cells, specific due outlets. to their This small phenomenon size, flow in can the becentral naturally channel observed whereas in blood white capillaries blood cells wherein flow in thered periphery. blood cells, due to their small size, flow in the centralYamamura channel whereas et al. proposed white blood a device cells for flow efficiently in the periphery. screening individual active B cells from an array of lymphocytesYamamura andet al. retrieving proposed targeted a devic cellse for (Figure efficiently 19)[152 screening,153]. Jin indivi et al.dual proposed active a modifiedB cells from design an arraywith microof lymphocytes array chip and for retrieving screening targeted and separating cells (Figure individual 19) [152,153]. cells that Jin secrete et al. proposed targeted a antibodies. modified designThis device with usesmicro the array principle chip of for highly screening sensitive and and sepa highlyrating specific individual enzyme cells linked that immunospot secrete targeted assay antibodies.(ELISPOT) forThis the device detection uses of the antibody principle secretion. of highly The chipsensitive has an and array highly of hollow specific sites enzyme with optimized linked immunospotdimensions to assay capture (ELISPOT) individual for the lymphocytes. detection of The antibody surface secretion. of the chip The is chip coated has with an array immunoglobin of hollow sitesspecific with antibody optimized (IgG-Ab). dimensions These to IgG-Ab capture trap individu the antibodiesal lymphocytes. secreted The by surface each individual of the chip cell is aroundcoated withthe trapping immunoglobin sites. On specific addition antibody of targeted (IgG-Ab). antibody These specific IgG-Ab fluorochrome trap the antibodies tagged antigens, secreted doughnut by each individualshaped fluorescent cell around signals the trapping can be observedsites. On onaddi thetion chip. of targeted Thus, targetedantibody individual specific fluorochrome cells can be taggedretrieved antigens, from the doughnut chip for shaped further fluorescent downstream sign analysisals can andbe observed application. on the This chip. device Thus, can targeted handle individual234,000 individual cells can cellsbe retrieved at a time, from thus the proving chip for to further be a high downstream throughput analysis cell detection and application. and retrieving This devicesystem can [154 handle]. 234,000 individual cells at a time, thus proving to be a high throughput cell detection and retrieving system [154]. Int.Int. J. J. Mol. Mol. Sci. Sci. 20182018, ,1919, ,x 3143 FOR PEER REVIEW 1818 of of 46 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 18 of 46

Figure 19. Schematic representation of the micro array-based detection and retrieval of targeted FigureFigure 19.19. SchematicSchematic representation representation of the of micro the micro array-based array-based detection detection and retrieval and retrieval of targeted of antibody secreting cells and downstream analysis. Reprinted with permission from [154]. Copyright targetedantibody antibody secreting secreting cells and cells down andstream downstream analysis. analysis.Reprinted Reprinted with permission with permission from [154]. from Copyright [154]. (2009) Springer Nature. Copyright(2009) Springer (2009) Nature. Springer Nature.

DevelopmentDevelopment of of antibodies antibodies for for numerous numerous diseases diseases obtained obtained from from B cells B cells is an is anupcoming upcoming field field in Development of antibodies for numerous diseases obtained from B cells is an upcoming field in therapeuticsin therapeutics study. study. The The antibodies antibodies for forparticular particular diseases diseases can can be obtained be obtained either either by byusing using media media of therapeutics study. The antibodies for particular diseases can be obtained either by using media of culturedof cultured B cells B cells or by or gene by gene cloning. cloning. However, However, this is this an isexpensive an expensive and laborious and laborious technique. technique. Next cultured B cells or by gene cloning. However, this is an expensive and laborious technique. Next generationNext generation sequencing sequencing (NGS) (NGS) is a us iseful a useful technique technique wherein wherein millions millions of B cells’ of B cells’ paired paired repertoires repertoires are generation sequencing (NGS) is a useful technique wherein millions of B cells’ paired repertoires are profiled.are profiled. However, However, it suffers it suffersfrom low from screening low screening throughput. throughput. In a recent Inadvancement, a recent advancement, Rajan et al. profiled. However, it suffers from low screening throughput. In a recent advancement, Rajan et al. [102]Rajan demonstrated et al. [102] demonstrated the use of thedroplet use ofmicrofluid droplet microfluidicsics for high forthroughput high throughput screening screening of paired of [102] demonstrated the use of droplet microfluidics for high throughput screening of paired repertoires.paired repertoires. Individual Individual B cells are B cells captured are captured in micro in droplets micro droplets along alongwith the with reagent. the reagent. This allows This allows high repertoires. Individual B cells are captured in micro droplets along with the reagent. This allows high throughputhigh throughput screening, screening, phage phage display display and NGS. and NGS. throughput screening, phage display and NGS.

3.2.3.3.2.3. Micro Micro Vortex Vortex Based Cell Isolation 3.2.3. Micro Vortex Based Cell Isolation StottStott et et al. al. [155] [155 showed] showed the the empl employmentoyment of microvortices of microvortices to isolat to isolatee rare cells rare from cells fluids. from fluids.They Stott et al. [155] showed the employment of microvortices to isolate rare cells from fluids. They extendedThey extended the technique the technique of mixing of mixing solutions solutions by generating by generating microv microvorticesortices in fluidic in fluidic channels channels via extended the technique of mixing solutions by generating microvortices in fluidic channels via transversevia transverse flows flows introduced introduced by Stroock by Stroock et al. et[156] al. [to156 capture] to capture CTCs CTCs from whole from whole blood. blood. Stott et Stott al. were et al. transverse flows introduced by Stroock et al. [156] to capture CTCs from whole blood. Stott et al. were ablewere to able detect to rare detect clusters rare clusters of 4–12 cells of 4–12 from cells input from blood, input which blood, were which not detected were not in detected the traditional in the able to detect rare clusters of 4–12 cells from input blood, which were not detected in the traditional CTCtraditional chip [80]. CTC Khojah chip [ 80et]. al. Khojah [103] demonstrated et al. [103] demonstrated the capture theof different capture ofsizes different of cells sizes by tuning of cells the by CTC chip [80]. Khojah et al. [103] demonstrated the capture of different sizes of cells by tuning the fluidtuning flow the properties fluid flow on properties chips having on chips the havingsame geometry the same under geometry all cell under inputs all cell(Figure inputs 20). (Figure 20). fluid flow properties on chips having the same geometry under all cell inputs (Figure 20).

FigureFigure 20. 20. SchematicSchematic diagram diagram of of the the device device proposed proposed by by Khojah Khojah et et al. al. with with a a micro-cavity micro-cavity 1 1 cm cm from from Figure 20. Schematic diagram of the device proposed by Khojah et al. with a micro-cavity 1 cm from thethe inlet. inlet. The The microvortex microvortex forms forms in in the the cavity cavity at at high high Reynolds Reynolds nu number.mber. Reproduced Reproduced from from [103] [103] with with the inlet. The microvortex forms in the cavity at high Reynolds number. Reproduced from [103] with permissionpermission of of The The Royal Royal Society Society of Chemistry. permission of The Royal Society of Chemistry.

TrappingTrapping reservoirs ofof thethe samesame height height but but different different aspect aspect ratios ratios were were placed placed at aat distance a distance of 1of cm 1 Trapping reservoirs of the same height but different aspect ratios were placed at a distance of 1 cmfrom from the the inlet inlet of theof the rectangular rectangular flow flow channel. channel. They They defined defined three three phases phases of flow,of flow, each each varying varying in thein cm from the inlet of the rectangular flow channel. They defined three phases of flow, each≤ varying in therange range of Reynolds of Reynolds number number of the of flow the flow in the in main the main channel. channel. In phase In phase I, corresponding I, corresponding to 100 to Re100 < ≤ 175Re , the range of Reynolds number of the flow in the main channel. In phase I, corresponding to 100 ≤ Re

4. SCT for Therapeutics Development Specific gene function studies within a single-cell and studies on gene response to drug molecules help scientists to understand the heterogeneity of a similar population of cells [157]. Many of these studies rely on introducing genes into cells. Several gene delivery methods have been developed, such as viral transduction, calcium phosphate precipitation, liposome, microinjection, sonoporation, electroporation, etc., [2,158–164]. Although none of these approaches offer all of the desired results at once, physical delivery techniques are advantageous in terms of high efficiency, high cell viability, better control, diverse cargo delivery capability, selectivity, cost-effectiveness, and reproducibility [27]. Viral transduction is a popular method used for the delivery of macromolecules. More than 90% of clinical trials being carried out are via viral transduction methods. The desired genetic material is incorporated in the genetic material of the viral vector. The viral vector is modified to suppress the disease-causing genes. This method is cell specific. The major advantage of viral transduction is that the viruses have natural mechanisms to overcome the cell membrane barrier of host cells. However, we cannot control the cargo dosage besides virus has limited genetic material carrying capability. Moreover, the viral transduction approach has immunogenic response issues and process customization is required after changing cargo and cell types [158]. In this section, we describe some of the recent techniques for therapeutics (Table3).

Table 3. Single-Cell therapeutic techniques.

Single-Cell S. No. Therapeutic Features Advantages Disadvantages References Techniques Using Immunogenic effects, Genetically engineered Promising intracellular limitation in genetic 1 Viral vector viruses are used for uptake, targeted [158] material carrying transfection. delivery. capacity. Low cell viability, Use of electric field for Cost effective, high 2 Electroporation formation of [158,165] making cell pores. delivery efficiency. irreversible pore. Use of laser for creating Contactless delivery Low throughput for 3 Optoporation transient cell method, high [166,167] single cell delivery. membrane pores. transfection efficiency. Sometimes there is a Transient membrane compromise between pores are formed by High cell viability, high 4 Mechanoporation high transfection [168–171] applying mechanical transfection efficiency. efficiency and high forces on cells. cell viability.

4.1. Single-Cell Electroporation Electroporation is a physical method used to deliver cargo into cells by creating nanopores in the cell membrane. Upon the application of an externally applied electric field, the cell membrane deforms creating membrane nanopores, when the applied voltage exceeds the cell membrane threshold voltage [172]. Single-cell electroporation was first demonstrated in 1998 when two carbon microelectrodes were placed in close proximity to a cell and an electric potential was applied Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 20 of 46 Int. J. Mol. Sci. 2018, 19, 3143 20 of 47 microelectrodes were placed in close proximity to a cell and an electric potential was applied between the electrodes; the cell membrane was deformed and molecules entered into the cell through diffusion between the electrodes; the cell membrane was deformed and molecules entered into the cell through [173]. Several other approaches to single-cell electroporation have been demonstrated using diffusion [173]. Several other approaches to single-cell electroporation have been demonstrated using micropipettes [174], microfabricated devices [175] and microfluidics [176]. Longo et al. through their micropipettes [174], microfabricated devices [175] and microfluidics [176]. Longo et al. through work created stable mammalian cell lines by transfecting them with desired cargo using their work created stable mammalian cell lines by transfecting them with desired cargo using electroporation [165]. Potter et al. demonstrated mammalian cell electroporation for in-vivo gene electroporation [165]. Potter et al. demonstrated mammalian cell electroporation for in-vivo gene therapy in cancer treatment and DNA vaccination [177]. Even though these methods offer advantages therapy in cancer treatment and DNA vaccination [177]. Even though these methods offer advantages such as high throughput and high efficiency, their application is still limited due to low cell viability such as high throughput and high efficiency, their application is still limited due to low cell [178,179]. viability [178,179]. With a view to overcoming the low cell viability issue, Kang et al. [180] developed a technique With a view to overcoming the low cell viability issue, Kang et al. [180] developed a technique known as nano fountain probe electroporation. Figure 21 schematically illustrates the nano fountain known as nano fountain probe electroporation. Figure 21 schematically illustrates the nano fountain probe approach. probe approach. The chip is fabricated incorporating a microfluidic cargo flow channel into a cantilever with its The chip is fabricated incorporating a microfluidic cargo flow channel into a cantilever with its opening at the tip. The chip has 12 microfluidic drug tube-embedded cantilevers, all connected to a opening at the tip. The chip has 12 microfluidic drug tube-embedded cantilevers, all connected to a common micro-reservoir. An electrical connection between the conductive wire and the conductive common micro-reservoir. An electrical connection between the conductive wire and the conductive cell substrate generates a concentrated localized electric field at the individual cell site, creating a cell substrate generates a concentrated localized electric field at the individual cell site, creating a pore pore in its membrane. A drug can then enter into the cell through the capillary effect, or a micropump in its membrane. A drug can then enter into the cell through the capillary effect, or a micropump can can be used to pump the drug into the cell. This gives better temporal resolution on the diffusive be used to pump the drug into the cell. This gives better temporal resolution on the diffusive drug drug entry. Such a system accessorized with a micromanipulator or an AFM can deliver a drug to a entry. Such a system accessorized with a micromanipulator or an AFM can deliver a drug to a precise precise location within the single cell and multiple single cells can be efficiently transfected as per location within the single cell and multiple single cells can be efficiently transfected as per requirement requirement within the shorter time range. Since the inception of nano fountain probe single-cell within the shorter time range. Since the inception of nano fountain probe single-cell electroporation, electroporation, the voltage requirements needed to realize a successful result have been reduced thefrom voltage kilovolts requirements to only a needed few volts. to realize Use aof successful a micro-reservoir result have prevents been reduced overconsumption from kilovolts of to drug only amolecules few volts. and Use the of asystem micro-reservoir can be parallelized prevents overconsumption for high throughput of drug applications. molecules andDrawbacks the system of this can betechnique parallelized include for highthe requirement throughput applications.of a conductive Drawbacks fluid for ofdelivery, this technique cell culture, include and the limitations requirement in ofdelivering a conductive large fluidcargo for into delivery, the cell. cell culture, and limitations in delivering large cargo into the cell.

Figure 21.21. SchematicSchematic representation representation of of the the packed packed nano nano fountain probe (NFP) for single-cell electroporation. Reprinted with permission from [180]. [180]. Copyright Copyright (2013) (2013) Amer Americanican Chemical Chemical Society. Society.

Santra et al.al. proposed proposed a a device device for for selective and localized single cell nano electroporationelectroporation (LSCNEP). ThisThis device consists of electrodes having 500 nm gap, fabricated on a glass chip to reduce the affected cellcell membranemembrane areaarea andand ensureensure singlesingle cellcell electroporation.electroporation. An insulator acting as thethe passivation layer layer avoids avoids direct direct cell cell contact contact with with the theelectrodes. electrodes. This reduces This reduces any heating any heating effect as effect well as wellcreation as creation of any ofharmful any harmful ions during ions during the proce thess. process. The cell The is cellseeded is seeded on top on of top the of device. the device. On Onapplication application of suitable of suitable voltage, voltage, an anintense intense electric electric field field acts acts on on the the local local region region of of the the cell cell membrane as shown in Figure 22[ [162].162]. The The device device is is capable capable of of achieving achieving high high throughput, throughput, precise precise drug delivery controllability, andand highhigh cellcell viabilityviability (98%)(98%) [[181].181]. Int. J. Mol. Sci. 2018, 19, 3143 21 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 21 of 46

FigureFigure 22. SchematicSchematic representation representation of of localized localized single single cell cell nano nano electropor electroporationation (LSCNEP) (LSCNEP) chip. chip. ReproducedReproduced from [162] [162] with the permission of AIP Publishing.

4.2.4.2. Optoporation Optoporation

4.2.1.4.2.1. Bulk Bulk Optoporation Optoporation OneOne of of the most promising emerging technolo technologiesgies for for intracellular intracellular delivery delivery is is photo photo or or optoporation,optoporation, where where a a laser laser is is focused focused onto onto a a cell cell membrane in in the the presence or absence of metallic nanoparticlesnanoparticles to to produce produce micro micro or nanobubbles, which which disrupt disrupt th thee cell cell membrane and and produce transienttransient membrane membrane pores pores to deliver molecules into cellscells [[182].182]. Membrane Membrane permeability permeability can can be be increasedincreased either either by by using using a tightly a tightly focused focused laser laser beam beam (pulsed (pulsed or continuous or continuous wave laser) wave or laser) by a broad or by lasera broad beam laser in combination beam in combination with sensitizing with sensitizing nanoparticles nanoparticles [183]. In 1984, [183 Tsukakoshi]. In 1984, et Tsukakoshi al. reported et an al. optoporationreported an optoporation technique to technique deliver membrane to deliver membrane impermeable impermeable molecules molecules into a cell into by a creating cell by creating pores usingpores a using pulsed a pulsedlaser [184]. laser Nikolskaya [184]. Nikolskaya et al. used et a al. continuous used a continuous laser to deliver laser fluorescent to deliver fluorescent molecules inmolecules neonatal in rat neonatal cardiac rat cells. cardiac Thiscells. study This demonstrates study demonstrates high target high specific target delivery specificdelivery and high and spatial high resolutionspatial resolution enabled enabled by optoporation by optoporation [185]. [ 185Andr]. Andrewew et al. et al.used used femtosecond femtosecond pulsed pulsed laser laser for for optoporatingoptoporating Chinese Chinese hamster hamster ovarian ovarian cells cells [186]. [186]. Use Use of of pulsed pulsed lasers lasers over over continuous continuous lasers lasers for for optoporationoptoporation gives gives better energy efficiency efficiency and reduces cell death. InIn photothermal photothermal delivery, delivery, metal metal nanoparticles nanoparticles are are used to create Localized Surface Plasmon ResonanceResonance (LSPR). (LSPR). The The laser laser electric electric field field generated generated is enhanced is enhanced due due to the to thelightening-rod lightening-rod effect. effect. As aAs result, a result, plasmonic plasmonic bubbles bubbles are created are created in the insurrounding the surrounding medium, medium, which generates which generates shock waves. shock Thesewaves. shock These waves shock disrupt waves the disrupt cell membrane the cell membrane to create transient to create pores transient [31]. Bastein pores [31 et]. al. Bastein studied et the al. visiblestudied and the visiblenear-infrared and near-infrared laser wavelengths laser wavelengths and their effects and their on effectsintracellular on intracellular delivery and delivery cell viabilityand cell after viability exposing after exposingmammalian mammalian cells. The study cells. proved The study that proved the use thatof near-infrared the use of near-infrared wavelength canwavelength achieve better can achieve cell viability better cell[187]. viability Ranhua [187 Xiong]. Ranhua et al. showed Xiong et that al. showedthe optoporation that the optoporation efficiency in presenceefficiency of in metal presence particles of metal is higher particles as compared is higher as to compared direct laser to exposure direct laser for exposure live Hela for cells live [188]. Hela However,cells [188]. nanoparticle-mediated However, nanoparticle-mediated intracellular intracellular delivery deliveryis limited is limitedto certain to certain sizes sizesand andtypes types of molecules,of molecules, and and is only is only suitable suitable for for specific specific cell-t cell-typesypes [189]. [189 ].All All these these approaches approaches come come under under bulk bulk levellevel optical optical transfection. transfection. 4.2.2. Focused Laser Beam Based Single-Cell Optoporation 4.2.2. Focused Laser Beam Based Single-Cell Optoporation Waleed et al. reported single cell transfection of MCF-7 cells with pAcGFP1-C1 plasmid coated Waleed et al. reported single cell transfection of MCF-7 cells with pAcGFP1-C1 plasmid coated 1 µm amino-based polystyrene microparticles using a femtosecond laser (800 nm) in combination 1 µm amino-based polystyrene microparticles using a femtosecond laser (800 nm) in combination with optical tweezers (1064 nm) (Figure 23). This technique offered control over the amount of with optical tweezers (1064 nm) (Figure 23). This technique offered control over the amount of plasmid that is inserted into a cell, high selectivity, and aseptic conditions due to the non-contact plasmid that is inserted into a cell, high selectivity, and aseptic conditions due to the non-contact nature of the technique. The main limitations of this technique are the low transfection efficiency of nature of the technique. The main limitations of this technique are the low transfection efficiency of 12.7% and the low transfection frequency of 20 cells per hour [166], which makes it not feasible for 12.7% and the low transfection frequency of 20 cells per hour [166], which makes it not feasible for practical applications. practical applications. Int. J. Mol. Sci. 2018, 19, 3143 22 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 22 of 46 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 22 of 46

(a) (b) (a) (b) Figure 23. Schematic representation of particle intracellular delivery. (a) 1064 nm laser (blue) is used FigureFigure 23. 23. SchematicSchematic representation ofof particleparticle intracellularintracellular delivery. delivery. (a ()a 1064) 1064 nm nm laser laser (blue) (blue) is is used used as as an optical tweezer to trap particle. (b) An 800-nm femtosecond laser (yellow) is used as a asan an optical optical tweezer tweezer to trap to particle.trap particle. (b) An (b 800-nm) An 800-nm femtosecond femtosecond laser (yellow) laser is(yellow) used as is a puncturingused as a puncturing laser to create pore on cell membrane. Redrawn from [166]. puncturinglaser to create laser pore to create on cell pore membrane. on cell membrane. Redrawn from Redrawn [166]. from [166]. 4.2.3. Metal Nanostructure Assisted Single-Cell Optoporation 4.2.3.4.2.3. Metal Metal Nanostructure Nanostructure Assisted Assisted Single-Cell Single-Cell Optoporation Optoporation Wu et al. reported another single cell transfection technique based on metal light interaction WuWu et al. reported reported another another single single cell cell transfec transfectiontion technique technique based based on on metal metal light interaction based nano bubble, which created shear stress on cell membrane (Figure 24). They fabricated a nano basedbased nano nano bubble, bubble, which which created created shear shear stress stress on ce cellll membrane membrane (Figure 24).24). They They fabricated fabricated a a nano nano blade by depositing 100-nm titanium on glass micropipette. This nano blade is held near the cell bladeblade by by depositing 100-nm 100-nm titanium titanium on on glass micr micropipette.opipette. This This nano nano blade blade is is held held near near the the cell cell surface. Pulsed laser is irradiated on the titanium nano structure. As a result of LSPR, micro bubbles surface.surface. Pulsed Pulsed laser laser is is irradiated irradiated on on the the titanium titanium nano nano structure. structure. As As a a result result of of LSPR, LSPR, micro micro bubbles bubbles are created, which disrupt the cell membrane to create transient pores [190]. Using this technique, areare created, whichwhich disruptdisrupt the the cell cell membrane membrane to to create create transient transient pores pores [190 [190].]. Using Using this this technique, technique, they they pumped the cargo into the cell through a micropipette with 46% transfection efficiency and 90% theypumped pumped the cargo the cargo into into the cellthe throughcell through a micropipette a micropipette with with 46% 46% transfection transfection efficiency efficiency and and 90% 90% cell cell viability [167]. cellviability viability [167 [167].].

Figure 24. Schematic representation of the Nano blade. The figure depicts the process of cell FigureFigure 24.24. SchematicSchematic representation representation of theof the Nano Nano blade. blad Thee. figure The depictsfigure depicts the process the ofprocess cell membrane of cell poremembrane formation pore by formation creating microby creating bubbles. micro Redrawn bubbles. from Redrawn [190]. from [190]. membrane pore formation by creating micro bubbles. Redrawn from [190]. 4.3.4.3. Single-CellSingle-Cell MechanoporationMechanoporation 4.3. Single-Cell Mechanoporation ThisThis techniquetechnique usesuses physical physical force force to to penetrate penetrate the the cell cell membrane membrane and and deliver deliver the the drug drug inside inside the This technique uses physical force to penetrate the cell membrane and deliver the drug inside cells.the cells. Microinjection Microinjection is an is example an example of mechanoporation. of mechanoporation. A microneedle A microneedle is used is used to inject to inject the drug the drug into the cells. Microinjection is an example of mechanoporation. A microneedle is used to inject the drug theinto cytosol. the cytosol. A micromanipulator A micromanipulator is used is used to hold to thehold microneedle the microneedle and control and control the pressure the pressure for cargo for into the cytosol. A micromanipulator is used to hold the microneedle and control the pressure for cargo delivery. This technique allows high delivery efficiency and a throughput of about 1000 cells cargo delivery. This technique allows high delivery efficiency and a throughput of about 1000 cells Int. J. Mol. Sci. 2018, 19, 3143 23 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 23 of 46 delivery.per hour. ThisThis techniquesystem is further allows highupgraded delivery in the efficiency development and a throughput of microfluidic of about devices 1000 with cells stationary per hour. Thisneedle system probes is and further flowing upgraded cells to in make the development the system more of microfluidic robust and increase devices withthroughput stationary [27]. needle Thus, probeshollow andengineered flowing probes cells to such make as the hollow system micro-cap more robustillary andarrays increase [168,169], throughput hollow nanoneedle [27]. Thus, hollow arrays engineered[191] and carbon probes nanosyring such as hollowe arrays micro-capillary [192] have been arrays fabricated [168, 169on the], hollow cellular nanoneedle scale. To achieve arrays better [191] andpenetration carbon nanosyringeof the cell membrane arrays [192 and] havesuccessive been fabricatedcellular injection, on the cellularthe wall scale.thickness To achieveof the hollow better penetrationprobes is reduced. of the cell Limited membrane efforts and have successive been ma cellularde to develop injection, a microprobe the wall thickness array for of thesingle-cell hollow probesmanipulation. is reduced. In particular, Limited to efforts reduce have the stress been madeconcentration to develop at the a cell, microprobe Shibata et array al. [193] for single-cellfabricated manipulation.a hollow microprobe In particular, array to with reduce hemispherical the stress concentration tips (Figure at 25). the cell,This Shibatadevice etis al.a microarray [193] fabricated chip, a hollowdesigned microprobe for high-throughput array with hemispherical intracellular tips delive (Figurery 25 of). Thissingle-cells device is that a microarray even allows chip, designedfor the formanipulation high-throughput of different intracellular types of deliverysingle-cells of in single-cells 3-D and parallel that even formats. allows For for fabrication the manipulation simplicity, of differentonly a single types photomask of single-cells was inused 3-D to and design parallel the formats.probes in For previous fabrication studies simplicity, [194]. The only fabrication a single photomaskprocess established was used hitherto to design lacks the probesapproaches in previous for monitoring studies [194 the]. inner The fabricationdiameter, outer process diameter established and hithertoa wall thickness lacks approaches of the probes. for monitoring It is essential the to inner improve diameter, the fabrication outer diameter process and to a control wall thickness the probe of thedimensions probes. Itand is essentialto optimize to improvethe structur thees fabrication used for single-cell process to manipulation. control the probe dimensions and to optimize the structures used for single-cell manipulation.

FigureFigure 25.25. OverviewOverview ofof thethe systemsystem designed by Shibata et al. “Reprinted with with permission permission from from [193]”. [193]”.

Recently, devicesdevices that usedused constrictionsconstrictions to applyapply physical stresses to cells for enablingenabling drug delivery intointo cells cells have have gained gained prominence prominence in the in field the offield mechanoporation. of mechanoporation. Sharei etSharei al. [170 et] proposedal. [170] theproposed first constrictions-based the first constrictions-ba devicesed for drugdevice delivery, for drug which delivery, forces which cells to forces pass throughcells to pass constrictions through inconstrictions series or parallel, in series causing or parallel, transient causing pores transi to developent pores in to the develop cell membranes in the cell duringmembranes this processduring (Figurethis process 26). The(Figure fluid 26). surrounding The fluid surrounding the cells are loaded the cells with are drugs,loaded which with drugs, diffuse which into the diffuse cells throughinto the thesecells through transient these pores. transient This device pores. showed This device a very show highed throughput a very high rate throughput of 20,000 cells/srate of 20,000 and delivery cells/s efficiencyand delivery between efficiency 70–90%. between Szeto 70– et90%. al. [171 Szeto] used et al. the [171] commercial used the Cell commercial Squeeze Cell platform, Squeeze based platform, on the devicebased on described the device by described Sharei et al.,by toSharei verify et theal., to delivery verify the of drugs delivery directly of drugs into directly the cytosol intoof the cells cytosol and notof cells endosomal and not compartmentsendosomal compartments through various through experiments. various experiments. Szeto et al. also Szeto conducted et al. also experiments conducted toexperiments optimize theto optimize device parameters the device forparameters maximizing for maximizing cell viability cell and viability delivery and efficiency. delivery Theyefficiency. used B-cellsThey used for theirB-cells experiments for their experiments and reported and cell reported viability cell above viability 95% above after 95% treatment after treatment with theplatform with the alongplatform with along more with than more 25-fold than increase 25-fold in increase internalization in internalization of desired of particles desired into particles the B-cells into the compared B-cells tocompared control conditions.to control conditions. Int. J. Mol. Sci. 2018, 19, 3143 24 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 24 of 46

Figure 26.26. (A(A) )Mechanism Mechanism of of formation formation of of transient transient pores pores in cells on passing through thethe microconstrictions in the device designed designed by by Sharei Sharei et et al.; al.; (B (B) )Image Image of of the the finished finished device device with with a azoomed-in zoomed-in view view showing showing the the cell cell flow flow ch channelsannels with with the the mi microconstrictions.croconstrictions. ( C)) Image of the devicedevice packaging.packaging. “Permission toto reprintreprint obtainedobtained fromfrom PNASPNAS [[170]”.170]”.

5. SCT Analytical Strategies Substantial progressprogress in in nanotechnology nanotechnology over over the last the two last decades two decades has greatly has improved greatly sensitivityimproved ofsensitivity analysis of at analysis an individual at an individual cell level. cell Here, level. we Here, present we some present of thesome recent of the methods recent reportedmethods forreported analyzing for analyzing a single-cell, a single-cell, including including cell tracking cell software, tracking alongsoftware, with along their workingwith their principles. working Theprinciples. design The and design application and ofapplication several devices of several that devices are highly that effective are highly in analyzingeffective in intracellular analyzing moleculesintracellular and molecules cell growth and rates cell growth will also rates be discussed will also (Tablebe discussed4). (Table 4).

Table 4.4. Single-cellSingle-cell analysisanalysis techniques.techniques.

Single-cellSingle-cell Analysis Analysis S No. Features References References TechniquesTechniques 4D recording4D recording AllowsAllows tracking tracking of targeted of targeted cell in cell X, Yinand X, Y Z and directions Z directions along withalong time. Softwarewith time. controlled motorized stage. 1 Single-cell differentiation BaSiC software allows detection of transcription factors as the stem cell [27,195] Software controlled motorized stage. 1 Single-cell differentiation undergoes differentiation. [27,195] W-4PiSMSNBaSiC software allows imagingallows detection of cell cross of sectiontranscription to view factors cell organelles as the based on high-resolutionstem cell undergoes fluorescence differentiation. microscopy. W-4PiSMSN allows imaging of cell cross section to view cell SMR Cellorganelles mass and growthbased on rate high-resolution can be quantified. fluorescence microscopy. 2 Single-cell growth QuantificationSMR based on the resonance of suspended cantilever before and [196] afterCell cell mass growth and cycle. growth rate can be quantified. 2 Single-cell growth Low-costQuantification technique. based on the resonance of suspended cantilever [196] Cell membrane potential Thebefore localized and potential after cell of growth and within cycle. a cell can be measured. 3 [197] measurement UsefulLow-cost in the studytechnique. of neurons. Cell membrane potential ThisThe is a localized live cell extraction potential technique.of and within a cell can be measured. 43 Intracellular access [197][198 ] measurement IntracellularUseful in contents the study can of be neurons. extracted over the cell cycle for analysis. StudyThis of is structure, a live cell function, extraction and technique. expression of cell genome/transcriptome 4 Intracellular access (mRNA)Intracellular in phenotypically contents can varying be extracted cells. over the cell cycle for [198] Genomics and 5 The transcriptomic study can give connecting link between the genetic [199] Transcriptomics analysis. makeupStudy and of phenotypicstructure, function, characteristics and expression of cells. of cell Uses Whole genome/transcriptome amplification (WGA/WTA) for NGS. Genomics and genome/transcriptome (mRNA) in phenotypically varying cells. 5 [199] Transcriptomics The transcriptomic study can give connecting link between the genetic makeup and phenotypic characteristics of cells. Int. J. Mol. Sci. 2018, 19, 3143 25 of 47

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 25 of 46 Table 4. Cont. Uses Whole genome/transcriptome amplification (WGA/WTA) Single-cell Analysis S No. Features References Techniques for NGS. It is the study of cellular protein structure and functions. It is the study of cellular protein structure and functions. [199] 66 ProteomicsProteomics UsesUses techniques techniques like masslike mass spectroscopy spectroscopy and antigen-antibodyand antigen-antibody reactions for [199] the studyreactions of proteins. for the study of proteins. UsefulUseful for analyzing for analyzing and separatingand separa cellularting cellular components components like DNA,like RNA, DNA, RNA, proteins, metabolites based on their mass. 77 MassMass Spectrometry Spectrometry proteins, metabolites based on their mass. [200,201][200, 201] CyTOFCyTOF useful useful for detecting for detecting single single cell surface cell surface and intracellularand intracellular markers. markers. 5.1. Better Visualization of Single-Cell Differentiation 5.1. Better Visualization of Single-Cell Differentiation MicroscopicMicroscopic monitoring monitoring of aof single-cell a single-cell from from a mass a ofmass cells of for cells the entirefor the duration entire duration of an experiment of an is challenging,experiment is partly challenging, because partly cells canbecause divide cells and can migrate divide rapidly.and migrat Toe recognize rapidly. To and recognize track targeted and track cells, onlytargeted a few microscopecells, only setups,a few establishedmicroscope undersetups, vibration-free established under conditions, vibration-free are available, conditions, which offerare a combinationavailable, which of high-resolution offer a combination micro-displacement of high-resolution stages micro-displacement with supporting software.stages with supporting software.A number of proprietary software packages are available that share a similar strategic approach to cell tracking.A number Inof proprietary this approach, software the region packages of are interest available is first that selected share a similar by the strategic operator approach using the software.to cell tracking. The algorithm In this approach, identifies the region center of of interest the cell is and first thus selected establishes by the operator a set of featuresusing the for trackingsoftware. the translocationThe algorithm of identifies the center the of acenter cell and of th itse morphologicalcell and thus establishes change along a set subsequent of features frames. for Thetracking micrometer-scale the translocation resolution of the stage center is repositioned of a cell and to its the morphological new location ofchange the cell along as soon subsequent as a large movementframes. The takes micrometer-scale place beyond theresolution starting stage position. is repo Assitioned longas to thethe Xnew–Y stagelocation facilitates of the cell micrometer as soon levelas adjustments,a large movement a cell takes can beplace reliably beyond tracked. the starting In addition position. to As such longX– asY stagethe X– displacement,Y stage facilitates most modernmicrometer systems level allow adjustments, for fine-tuning a cell ofcan the beZ-axis reliably to investigate tracked. In cells addition suspended to such above X–Y the stage focal plane.displacement, Sub-micron most adjustment modern systems of the allowZ-axis for allows fine-tuning for the of the selection Z-axis to of investigate vertical optical cells suspended slices, thus enablingabove spatiotemporalthe focal plane. Sub-micron 4D recording adjustment of cells for of rendering the Z-axis and allows modeling for the [selection27]. Figure of vertical27 schematically optical slices, thus enabling spatiotemporal 4D recording of cells for rendering and modeling [27]. Figure 27 illustrates the tracking of individual cells from a population of live cells. schematically illustrates the tracking of individual cells from a population of live cells.

FigureFigure 27. 27.Tracking Tracking of individualof individual cells cells from from a population a population of live of culturing live culturing cells usingcells ausing live-cell a live-cell imaging microscopicimaging microscopic platform with platform a high-resolution with a high-resolution micro-displacement micro-displacement motorized stage. motorized “Reprinted stage. with permission“Reprinted from with [202 permission]. Copyright from (2016) [202]. SpringerCopyright Nature”. (2016) Springer Nature”. Int. J. Mol. Sci. 2018, 19, 3143 26 of 47

A software named BaSiC has been used to observe obscure changes involved in cell development. It can successfully detect transcription factors used by cells during decisive developmental steps. Such detection requires prolonged tracking of cells with background alterations and shading, which is tedious using traditional tools. However, BaSiC has an inbuilt background alteration correction algorithm, which is an exceptional tool for the study of early-onset transcriptional factors and is especially useful in stem cell studies. One of the key features of BaSiC is the use of the L1 norm for error measurement, which leads to faster convergence, thus requiring fewer images for processing compared to other techniques. This is especially appreciable in situations where only a limited number of images are available. Using the L1 norm also allows for sparser solutions, thus accommodating outliers such as noise better than other techniques. BaSiC also uses a spatially constant baseline signal Bi to account for varying background in subsequent images. Using this tool, developers observed hematopoietic stem cell differentiation. The results could visibly differentiate between the up-regulation of a certain transcription factor in one of its mature cell lineages which remained dormant in its other type of mature cell lineage, providing information on decisive factors in cell development [195]. Another software that deals with the issue of uneven illumination without requiring any reference images is CIDRE (correction intensity distributions using regularized energy minimization), introduced by Kevin Smith et al. [203] A predecessor to BaSiC, CIDRE too uses both the flat-field and dark-field to stitch images. One of the major assumptions of CIDRE is that the input images are uncorrelated. CIDRE matches the performance of BaSiC for larger collection of images. CIDRE estimates the zero light or dark light term z and the illumination gain v at all points simultaneously using an energy minimization technique [204]. The method models distortions to images by the following equation:

I (x) − z(x) I(x) = 0 v(x) where I(x) refers to the true image and I0(x) refers to the image captured by the sensor. Since v and z are already determined by the method as described above, the true image is extracted using this equation.

5.2. Quantifying Single-Cell Growth The growth rate of a cell is governed by a combination of several factors. Even genetically identical cells can have different growth rates due to different combinations of intrinsic molecular noise and various deterministic behavioral programs [205–209]. Despite having important consequences for human health, this variation is less observable via population-based growth assays. Recently, a number of methods have been proposed for measuring single-cell growth. One of these techniques involves resonating MEMS structures where a low concentration cell suspension is allowed to flow through sealed microchannels. These microchannels are etched along the cantilever length. The cantilever itself is suspended in a vacuum cavity. The principle of the operation is a change in the natural frequency of the cantilever resonator on pumping the cell suspension through the microchannel (with respect to pumping suspension without cells). Gobin et al. used a suspended microchannel resonator (SMR) along with a picoliter scale microfluidic control to measure mass and determine the instantaneous growth rates of an individual cell [210]. Son et al. developed a microfluidic system, which can measure single-cell mass and cell-cycle progression simultaneously over multiple generations [211]. Another SMR chip designed by Burg et al. consists of a sealed microfluidic channel, which is placed inside a cantilever resonator. This cantilever is housed in an on-chip vacuum cavity. When a cell suspension flows through the interior of the cantilever, it transiently changes the cantilever’s resonant frequency in proportion to the mass of the cell. These methods measure the growth of a single-cell with high precision at the cost of throughput. To overcome this limitation, a serial array of such micro cantilevers containing intermediate delay channels was presented by Cermak et al. [196]. Figure 28 shows the SMR and its working principle. Int.Int. J.J. Mol.Mol. Sci.Sci.2018 2018,,19 19,, 3143x FOR PEER REVIEW 27 27 of of 47 46

FigureFigure 28.28.( (aa)) SeriesSeries of of SMRs SMRs (cantilever (cantilever mass mass sensors) sensors) separated separated by by delay delay channels; channels; (b ()b Rendering) Rendering of of a largea large channel channel serial serial SMR SMR array array device device showing showing delay delay channels channels and theand cantilevers the cantilevers (magnified (magnified in inset in micrograph).inset micrograph). Reprinted Reprinted with permission with permission from[ from196]. Copyright[196]. Copy (2016)right (2016) Springer Springer Nature. Nature.

The intermediate delay allows for the essential time-span required for cell division, which can The intermediate delay allows for the essential time-span required for cell division, which can be quantified in the microchannel embedded cantilever region. Thus, high precision using the single be quantified in the microchannel embedded cantilever region. Thus, high precision using the single suspended microchannel resonator (SMR) can be achieved. Growth rates of over 1 cells/min can be suspended microchannel resonator (SMR) can be achieved. Growth rates of over 1 cells/min can be obtained using this technique. Observation of variations in individual cell growth is a very important obtained using this technique. Observation of variations in individual cell growth is a very important aspect of cancer studies as well as degenerative disease research. This approach can give important aspect of cancer studies as well as degenerative disease research. This approach can give important information on drug efficacy in terms of cell growth [196]. information on drug efficacy in terms of cell growth [196]. An important factor along with growth rate quantification is cell cycle inheritance. An important factor along with growth rate quantification is cell cycle inheritance. Sandler et al. Sandler et al. [209] developed a model for cell cycle inheritance using microfluidic flow to deposit [209] developed a model for cell cycle inheritance using microfluidic flow to deposit them into them into microwells, observing them using time-lapse microscopy. They developed a ‘kicked cell microwells, observing them using time-lapse microscopy. They developed a ‘kicked cell cycle’ model cycle’ model to explain the correlation observed between cousin cells; there seemed to be close to to explain the correlation observed between cousin cells; there seemed to be close to zero correlation zero correlation between mother-daughter cells. The main feature in this model was the deterministic between mother-daughter cells. The main feature in this model was the deterministic influence of the influence of the circadian phase at birth on the cell cycle duration [212]. They were successfully able to circadian phase at birth on the cell cycle duration [212]. They were successfully able to validate the validate the observed correlations with their model, thus demonstrating the importance of a non-linear observed correlations with their model, thus demonstrating the importance of a non-linear process, process,such as suchthe circadian as the circadian clock [213], clock on [213 the], determination on the determination of cell cycle of cell variability. cycle variability. 5.3. Single-Cells in 3D View 5.3. Single-Cells in 3D View Vast technical improvements in microscopical techniques have been made over recent decades Vast technical improvements in microscopical techniques have been made over recent decades to increase the contrast between signal and background. High-resolution fluorescence microscopic to increase the contrast between signal and background. High-resolution fluorescence microscopic technologies are highly effective in understanding the molecular-level details that underpin living technologies are highly effective in understanding the molecular-level details that underpin living organism processes. The large spectral range of available fluorophores allows for simultaneous organism processes. The large spectral range of available fluorophores allows for simultaneous imaging of different cellular, subcellular, or molecular components. One important contribution to imaging of different cellular, subcellular, or molecular components. One important contribution to the development of SCT is whole cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), with the development of SCT is whole cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), with three-dimensional individual cell imaging capability [214]. W-4PiSMSN can provide 10–20 nm axial three-dimensional individual cell imaging capability [214]. W-4PiSMSN can provide 10–20 nm axial resolution in the volumetric reconstruction of 10 µm thick samples. Cell sections which previously resolution in the volumetric reconstruction of 10 µm thick samples. Cell sections which previously could only be visualized by electron microscopy could now be studied by W-4PiSMSN. This overcomes could only be visualized by electron microscopy could now be studied by W-4PiSMSN. This the electron microscopy associated drawback in terms of the ability to study the localization of overcomes the electron microscopy associated drawback in terms of the ability to study the proteins within a cell. The group successfully reconstructed Golgi complex-associated COPI vesicles, localization of proteins within a cell. The group successfully reconstructed Golgi complex-associated endoplasmic reticulum, nuclear pore complexes and cell cilia of COS-7 cells and icosahedra shaped COPI vesicles, endoplasmic reticulum, nuclear pore complexes and cell cilia of COS-7 cells and capsids in bacteriophages. icosahedra shaped capsids in bacteriophages.

Int. J. Mol. Sci. 2018, 19, 3143 28 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 28 of 46

5.4.5.4. Cell Cell Membrane Membrane Potential Potential Measurement Measurement ScientistsScientists often often strivestrive to study study the the membrane membrane potential potential change change occurring occurring within within individual individual cells cellsdue due to ion to ionexchange exchange performed performed during during cell signaling. cell signaling. Techniques Techniques such as such patch as clamp, patch planar clamp, patch planar patchclamp, clamp, and andtheir their subtypes subtypes have have been been used used to study to study ion ion exchange exchange during during intercellular intercellular signaling. signaling. However,However, such such techniques techniques are unable are unable to preserve to preserve the cellular the microenvironment cellular microenvironment during measurements. during Furthermore,measurements. artificial Furthermore, stimulations artifici needal stimulations to be applied need to theto be cell, appli typicallyed to the using cell, a micropipette,typically using prior a tomicropipette, measurement prior of its to response. measurement The of drawback its response. lies The in the drawback disturbance lies in produced the disturbance in the produced cytosol due toin dilution the cytosol with due micropipette to dilution contents. with micropipette In order contents. to overcome In order these to drawbacks, overcome these nanowire drawbacks, devices havenanowire been developed devices have to measurebeen developed intracellular to measure potential intracellular with nominal potential invasiveness. with nominal These invasiveness. devices can measureThese devices the potential can measure of one the individual potential cell of one at a individu time, asal well cell as at aa networktime, as well of cells as a simultaneously.network of cells simultaneously.One such device was developed by Liu et al. [197] (Figure 29); it employs an electric circuit model One such device was developed by Liu et al. [197] (Figure 29); it employs an electric circuit model of the electro neuronal system, which has multiple electrodes connected to a single cell. These electrodes of the electro neuronal system, which has multiple electrodes connected to a single cell. These can measure the intracellular potential at multiple locations within a single cell. Moreover, it has the electrodes can measure the intracellular potential at multiple locations within a single cell. Moreover, exceptional capability of uniquely controlling each individual electrode. it has the exceptional capability of uniquely controlling each individual electrode.

Figure 29. The electrical circuit model of the electro-neural system starts with the cell culture (yellow) Figure 29. The electrical circuit model of the electro-neural system starts with the cell culture (yellow) interfacing with the electrode, all the way to the read-out electronics represented by the amplifier interfacing with the electrode, all the way to the read-out electronics represented by the amplifier block block (green). Reprinted with permission from [197]. Copyright (2017) American Chemical Society. (green). Reprinted with permission from [197]. Copyright (2017) American Chemical Society. Each electrode is 10 µm in length and 200 nm in diameter. The electrodes are spaced at a distance Each electrode is 10 µm in length and 200 nm in diameter. The electrodes are spaced at a distance of 4 µm from each other. The fabrication process is uniquely adapted to avoid any electrochemical of 4 µm from each other. The fabrication process is uniquely adapted to avoid any electrochemical interactions between the cell and the chip at any undesirable points except for the electrode tip interactions between the cell and the chip at any undesirable points except for the electrode tip required required for cell contact. This allows precise control when obtaining a localized signal from for cell contact. This allows precise control when obtaining a localized signal from intracellular intracellular locations. Low contact resistance and high input impedance are achieved to avoid any locations.errors due Low to contactnoise interference. resistance andThis highnanowire input chip impedance has been are successfully achieved toused avoid to measure any errors sub- due tothreshold noise interference. postsynaptic This potential nanowire in human-induced chip has been pluripotent successfully stem used cell-derived to measure neurons sub-threshold in mouse postsynapticand rat primary potential neurons in human-inducedwith high signal to pluripotent noise ratio [197]. stem cell-derivedThe study wa neuronss performed in mouse on the same andrat primarycells in neuronsvitro over with a period high of signal six weeks to noise with ratioexcellent [197 neuronal]. The study activity was preserved performed in human-induced on the same cells inpluripotent vitro over astem period cell-derived of six weeks neurons. with The excellent chip was neuronal proven activitycapable preservedof recording in small human-induced quantity pluripotentneurotransmitter stem cell-derived variations with neurons. signal Theprecision chip from was provenan individual capable cell of in recording a network small of neuronal quantity neurotransmittercells. Obtaining such variations data is withvital to signal the study precision of pre-synaptic from an individual and post-synaptic cell in adisorders. network It of can neuronal also cells.be useful Obtaining in modeling such data any is changes vital to thein potential study of frequency pre-synaptic or magnitude and post-synaptic of diseased disorders. conditions It can over also benormal useful inconditions modeling [197]. any changes in potential frequency or magnitude of diseased conditions over normal conditions [197]. 5.5. Cytometry 5.5. Cytometry Several methods are available for single-cell analysis, including laser induced fluorescence, electrochemicalSeveral methods detection are available(ECD) or formass single-cell spectrometry. analysis, These including methods, laseraccompanied induced by fluorescence, capillary electrochemicalelectrophoresis, detection have attracted (ECD) great or mass attention spectrometry. for their Theseability methods,to analyze accompanied low concentrations by capillary of electrophoresis,biomolecules [215–217]. have attracted Zheng et greatal. developed attention a lactate for their sensor ability with a tonanoscale analyze optical low fiber concentrations to detect Int. J. Mol. Sci. 2018, 19, 3143 29 of 47 of biomoleculesInt. J. Mol. Sci. 2018 [215, 19, –x 217FOR]. PEER Zheng REVIEW et al. developed a lactate sensor with a nanoscale 29 of 46 optical fiber to detect extracellular lactate in cancer cells [218]. However, these methods are laborious extracellular lactate in cancer cells [218]. However, these methods are laborious in estimating the in estimating the fluorescence intensity outside and inside living cells. Matrix Assisted Laser fluorescence intensity outside and inside living cells. Matrix Assisted Laser Desorption/Ionization- Desorption/Ionization-Mass Spectrometry (MALDI-MS) analysis of single peptide-containing vesicles Mass Spectrometry (MALDI-MS) analysis of single peptide-containing vesicles was established by Li was establishedet al. [200], but by the Li etmolecular al. [200], weight but the of molecular the detected weight biomolecules of the detected was restricted biomolecules to 500 to was8000 restricted Da. to 500Electrochemical to 8000 Da. electrodes Electrochemical coupled with electrodes sampling coupled capillaries with for sampling sampling very capillaries low concentrations for sampling veryof low mammalian concentrations cells was of developed mammalian by Woods cells et was al. [ developed219]. By means by of Woods a capillary-microfluidic et al. [219]. By device, means of a capillary-microfluidicOmiatek et al. [220] device, describe Omiatek cell separation, et al. [220 lysis,] describe and electrochemically cell separation, measured lysis, and the electrochemically contents of measuredsingle thevesicles. contents of single vesicles. AnotherAnother technique technique developed developed byby WuWu etet al. [221] [221] is is capable capable of ofzeptomole-level zeptomole-level detection detection of of neurotransmittersneurotransmitters released released from singlefrom livingsingle cellsliving using cells nanocapillary using nanocapi electrophoreticllary electrophoretic electrochemical electrochemical (Nano-CEEC) chips. The chip is comprised of three units, a pair of targeting (Nano-CEEC) chips. The chip is comprised of three units, a pair of targeting electrodes for capturing electrodes for capturing single-cells, a dual asymmetry electrokinetic flow device for sample single-cells,collection, a dualpre-concentration asymmetry electrokinetic and separation, flow and device an amperometric for sample collection,sensor for pre-concentrationthe detection and and separation,analysis and of neurotransmitters. an amperometric sensorFigure for30 schematically the detection illustrates and analysis the ofnano-CEEC neurotransmitters. chip for living Figure 30 schematicallysingle-cell illustratesanalysis. the nano-CEEC chip for living single-cell analysis.

FigureFigure 30. 30.Schematic Schematic representation representation of of a aNano-CEEC Nano-CEEC chip chip designed designed for living for sing livingle-cell single-cell analysis. Step analysis. 1: (a) cell loading, capturing and culturing in the cell chamber; Step 2: (b1,b2) DAEKF (dual- Step 1: (a) cell loading, capturing and culturing in the cell chamber; Step 2: (b1,b2) DAEKF asymmetry electrokinetic flow) separation, including sample collection, nanoseparation and (dual-asymmetry electrokinetic flow) separation, including sample collection, nanoseparation and restacking in the nanochannel; Step 3: (c) amperometric detection by the EC detection electrodes; (d) restacking in the nanochannel; Step 3: (c) amperometric detection by the EC detection electrodes; Schematics showing the flow-field evolution for DAEKF electrophoresis manipulated by surface zeta (d) Schematicspotentials in showing the nanochannel the flow-field (solid arrows evolution repres forent DAEKF the analyte electrophoresis flow direction) manipulated Reproduced byfrom surface zeta[221] potentials with permission in the nanochannel of The Royal (solid Society. arrows represent the analyte flow direction) Reproduced from [221] with permission of The Royal Society. The entire process can be completed in 15 min, right from cell sampling to neurotransmitter detection.The entire These process devices can might be completed be potentially in applicable 15 min, right for disease from celldiagnosis. sampling to neurotransmitter detection.Cytometry These devices Time of might Flight be (CyTOF) potentially is an advanc applicableed flow for cytometry disease diagnosis. technique that overcomes the spectralCytometry overlap Time issue of Flight involved (CyTOF) in studying is an multiple advanced parameters flow cytometry with FACS. technique This technique that overcomes allows the spectralthe study overlap of around issue involved 42 parameters in studying simultaneously. multiple Heavy parameters metal isotopes with FACS. are first This attached technique to the allows the studyantibodies of around for trapping 42 parameters targeted cells simultaneously. [222]. Once the targeted Heavy cell metal antigens isotopes are detected are first by attachedantibodies, to the single cells are encapsulated in droplets by a nebulizer (Figure 31). These encapsulated cells are then antibodies for trapping targeted cells [222]. Once the targeted cell antigens are detected by antibodies, passed through an inductively coupled plasma (ICP), which vaporizes them into an ion cloud. The single cells are encapsulated in droplets by a nebulizer (Figure 31). These encapsulated cells are Quadrupole Time of Flight (qTOF) mass spectrometry technique is used to measure mass to charge thenratio, passed which through can anprovide inductively the spectra coupled for tagge plasmad metal (ICP), atoms which [223]. vaporizes Thus, themthe antibody-metal into an ion cloud. The Quadrupoleconjugation data Time along of with Flight the (qTOF) spectrum mass obtained spectrometry by qTOF can technique provide iscell used marker to measureanalysis. Kay mass to charge ratio, which can provide the spectra for tagged metal atoms [223]. Thus, the antibody-metal conjugation data along with the spectrum obtained by qTOF can provide cell marker analysis. Kay et al. Int. J. Mol. Sci. 2018, 19, 3143 30 of 47

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 30 of 46 reported analysis of natural killer cells by using the CyTOF technique. Huge diversity is observed in theet expression al. reported of analysis surface of and natural intracellular killer cells markers by using expressed the CyTOF by naturaltechnique. killer Huge cells diversity based is on the targetedobserved disease. in the Suchexpression variability of surface requires and intracellular a large number markers ofexpressed probes by for natural analysis, killer whichcells based is made on the targeted disease. Such variability requires a large number of probes for analysis, which is made possible by the CyTOF technique [201]. However, CyTOF has lower throughput (1000 cells/s) as possible by the CyTOF technique [201]. However, CyTOF has lower throughput (1000 cells/s) as comparedcompared to FACS. to FACS.

FigureFigure 31. Schematic 31. Schematic representation representation of of the theworking working prin principleciple of of CyTOF. CyTOF. Reprinted Reprinted with with permission permission fromfrom [224 ].[224].

ErikErik Gerdtsson Gerdtsson et al.et al. [225 [225]] showed showed that that thethe integrationintegration of of Imaging Imaging Mass Mass Cytometry Cytometry (IMC) (IMC) using using metal-labeled antibodies into the existing high definition single-cell analysis [226] allowed the CTC metal-labeled antibodies into the existing high definition single-cell analysis [226] allowed the CTC profiling to be extended to include lot more protein biomarkers. The IMC process involves the profiling to be extended to include lot more protein biomarkers. The IMC process involves the coupling of laser ablation with single-cell mass cytometry performed using the CyTOF technology coupling[227]. of They laser reported ablation a with multiplexing single-cell level mass of cytometry40 proteins, performed which can using be used the for CyTOF the generation technology of [227]. Theyinformative reported a multiplexingbiomarker panels. level Thus, of 40 it proteins,shows high which potential can befor used complete for the characterization generation of of informative blood biomarkersamples panels. and extraction Thus, it of shows crucial high biomarkers potential for forassessing complete the liquid characterization phase of cancer. of blood samples and extraction of crucial biomarkers for assessing the liquid phase of cancer. 5.6. Single Cell Sequencing 5.6. Single Cell Sequencing Next Generation sequencing (NGS) is a sequencing technology that enables the sequencing of Nextentire Generationhuman genome sequencing within a day. (NGS) In this is techniqu a sequencinge, the entire technology genome is that first enablesdivided into the millions sequencing of entireof fragments human genomeand sequenced within in a parallel. day. In The this data technique, obtained the from entire individual genome fragments is first dividedare then into millionsmapped of fragments to a reference and human sequenced genome in via parallel. bioinformatics The data analysis. obtained This fromprocess individual is repeated fragments multiple are times to get accurate data on any DNA mutation. NGS can also be used for extracting data from then mapped to a reference human genome via bioinformatics analysis. This process is repeated targeted genetic locations. It can be used for detecting novel diseases, as it interrogates each multipleindividual times fragment to get accurate without data any prior on any knowledge DNA mutation. of the gene NGS loci. NGS can alsohas better be used sensitivity for extracting towards data fromlow-frequency targeted genetic variants locations. due to its It canhighbe sequencing used for depth detecting [228]. novelNGS along diseases, with drop-seq as it interrogates technique each individualhas many fragment applications without in DNAseq, any prior RNAseq knowledge and ATAC of theseq gene (chromatin) loci. NGS of has multiple better individual sensitivity cells. towards low-frequencyIn RNAseq, variants mRNA is duereverse to itstranscribed high sequencing to DNA, wh depthich is [further228]. NGS used alongas the substrate with drop-seq for analysing technique has manythe cells’ applications state based on in DNAseq,RNA molecule RNAseq expression and ATACseqprofile. This (chromatin) can also be used ofmultiple for proteomic individual studies. cells. In RNAseq,Gawad mRNAet al. explained is reverse the transcribedsignificance of to single-c DNA, whichell genomic is further analysis used in understanding as the substrate intratumor for analysing the cells’cell heterogeneity. state based on Such RNA analysis molecule can expressionprovide dyna profile.mic data This on canthe alsoprocess be usedof cancer for proteomicdevelopment studies. [229]. Buenrostro et al. showed that analysis of the single cell epigenome could be done quick enough Gawad et al. explained the significance of single-cell genomic analysis in understanding intratumor to enable users to make better clinical decisions [230]. Some of the major technologies developed cell heterogeneity. Such analysis can provide dynamic data on the process of cancer development [229]. recently also include RNA-Seq to study gene expression in bulk tissues, specifically single-cell RNA- BuenrostroSeq done et by al. Wu showed et al. [231]. that This analysis single-cell of the techni singleque cell was epigenome shown to be couldquantitatively be done comparable quick enough to to enablemultiplexed users to make quantitative better clinical PCR (qPCR) decisions [232]. [230 ].Another Some ofadvancement the major technologies in this technology developed is the recently also include RNA-Seq to study gene expression in bulk tissues, specifically single-cell RNA-Seq done by Wu et al. [231]. This single-cell technique was shown to be quantitatively comparable to multiplexed Int. J. Mol. Sci. 2018, 19, 3143 31 of 47 quantitative PCR (qPCR) [232]. Another advancement in this technology is the development of highly multiplexedInt. J. Mol. single-cellSci. 2018, 19, xqPCR FOR PEER introduced REVIEW by Vanlnsberghe et al. This device integrates all the 31 stepsof 46 for qPCR, including cell detection, isolation, and lysis for the measurement of qPCR reactions of 200 single cells indevelopment parallel. The of highly data obtained multiplexed revealed single-cell cell-to-cell qPCR introduced heterogeneity by Va fornlnsberghe two cell et types al. This following device cell differentiationintegrates all [233 the]. steps The for major qPCR, difference including between cell detec RNA-seqtion, isolation, and and qPCR lysis is for that the themeasurement former allows of a broaderqPCR scope reactions of gene of 200 analysis single atcells lower in parallel. sensitivity, The data which obtained is the revealed opposite cell-to-cell for the qPCRheterogeneity technology. for two cell types following cell differentiation [233]. The major difference between RNA-seq and qPCR 5.7. Intracellularis that the former Access allows a broader scope of gene analysis at lower sensitivity, which is the opposite for the qPCR technology. The study of intracellular proteins and mRNA is a pivotal step in the study of pathogenesis. Many5.7. highly Intracellular sensitive Access single-cell mRNA and protein detection methods, such as those based on phenotypeThe heterogeneity study of intracellular and noise proteins in cellular and systems,mRNA is have a pivotal been step developed in the study [229, 231of pathogenesis.]. These methods rely onMany lysing highly the cellssensitive to extract single-cell the intracellularmRNA and prot contentsein detection for analysis. methods, This such limitation as those isbased particularly on problematicphenotype when heterogeneity studying dynamicand noise transformations. in cellular systems, To overcomehave been the deve limitation,loped [229,231]. many minimallyThese invasivemethods sampling rely on devices lysing the have cells been to extract designed the tointr accessacellular the contents interior for of analysis. cell matrices. This limitation One approach is insertedparticularly atomic forceproblematic microscope when tipsstudying through dynamic a cell transformations. surface to make To directovercome contact the limitation, with the cytoplasmmany of livingminimally cells with invasive high sampling positional devices accuracy. have Thisbeen methoddesigned was to access used the to quantifyinterior of mRNA cell matrices. expression One in a singleapproach living cell inserted without atomic causing force severemicroscope damage tips through to the cell a cell [232 surface]. Saha-Shah to make direct et al. contact used nano-pipettes with the cytoplasm of living cells with high positional accuracy. This method was used to quantify mRNA (diameters < 1 µm) to collect nano-liter volumes (<10 nL) of cell-matrix samples from single Allium Cepa expression in a single living cell without causing severe damage to the cell [232]. Saha-Shah et al. cells [233]. However, this technique may impact cell survival and limit cell studies to cells of relatively used nano-pipettes (diameters < 1 µm) to collect nano-liter volumes (<10 nL) of cell-matrix samples largefrom sizes. single A recent Allium development Cepa cells [233]. by CaoHowever, et al. this [198 technique] has studied may theimpact limitations cell survival involved and limit with cell lysing cells instudies order to to cells extract of relatively its contents. large sizes. A recent development by Cao et al. [198] has studied the Figurelimitations 32 showsinvolved the with schematic lysing cells representation in order to extract of nanostrawsits contents. and their operations. The chip has nanostrawsFigure 32 embedded shows the verticallyschematic representation in the cell culture of nanostraws substrate. and On their applying operations. a bulk The electricchip hasfield, nanoporesnanostraws are created embedded on cells.vertically The in intracellular the cell culture contents substrate. drain On out applying through a thebulk nanostraws electric field, into a buffernanopores medium byare diffusion.created on The cells. nanostraws The intracellular being 150contents nm indrain diameter out through can be the used nanostraws for small molecules,into a proteinsbuffer and medium mRNA by extraction diffusion. throughThe nanostraws nanopores being existing 150 nm for in longerdiameter durations can be used without for small cytosolic molecules, proteins and mRNA extraction through nanopores existing for longer durations without leakage. It provides a dynamic non-destructive intracellular sampling environment to study the same cytosolic leakage. It provides a dynamic non-destructive intracellular sampling environment to study cell or set of cells throughout its cell cycle [198]. the same cell or set of cells throughout its cell cycle [198].

FigureFigure 32. Design 32. Design and and operation operation of of the the NEX NEX (Nanostraw (Nanostraw Extraction) Extraction) sampling sampling system. system. (A) (TheA) Thesystem system consistsconsists of a polymerof a polymer membrane membrane with with protruding protruding 150-nm150-nm diameter diameter nanostraw nanostraw (NS) (NS) attached attached to the to the bottombottom of a cell-cultureof a cell-culture dish. dish. Sampling Sampling is is performed performed by temporarily temporarily electropor electroporatingating the thecells cells cultured cultured on the NS, allowing cellular content to diffuse through the NS and into the underlying extraction on the NS, allowing cellular content to diffuse through the NS and into the underlying extraction buffer buffer (pink). An aliquot of the buffer is then aspirated with a standard pipette and analyzed (pink). An aliquot of the buffer is then aspirated with a standard pipette and analyzed conventionally, conventionally, using fluorescence imaging, ELISA, or qPCR; (B) Schematic representation of the using fluorescence imaging, ELISA, or qPCR; (B) Schematic representation of the extraction process. extraction process. Permission to reprint obtained from PNAS [198]. Permission to reprint obtained from PNAS [198]. Int. J. Mol. Sci. 2018, 19, 3143 32 of 47

6. Development, Challenges, Applications and Future Prospects of Single-cell Technologies The entire article provides a compact outlook to the reader about the stages involved in SCT development for different cellular characterization, disease analysis, etc. Manipulation of multiple cells and their behavior are essential to understand the in-vivo environment to reduce possible errors. The corresponding tools are continuously being developed; such as optical tweezer (1988) [234], manual micropipette (2001) [235], automated micropipette (2016) [236–238], manual pump based parallel micropipette array (2015) [70], etc. The optical tweezer was first introduced to trap bacterial cells in 1988 using an infrared laser source [236]. A decade later, dielectrophoretic tweezers were used for cell handling within microfluidic channels [239–241]. In 2000 [242–245], optical tweezers showed wide applications in the study of physical properties of cell, protein and DNA interactions. However, dielectrophoretic tweezers did not show such rapid development. Techniques such as rapid electrokinetic patterning (REP, 2008) and optoelectrokinetic tweezers (OET,) emerged in 2008 and 2013 [246–248]. As compared to single optical trap, multiple optical traps from single laser source were demonstrated recently in 2017. Compared to the recent developments in these techniques, microfluidic-based microarray probe shows a lack of cell selection flexibility, although it has the capability to increase throughput. However, the technique of 3D optical traps has lower throughput and is more expensive than microfluidic technique. Nevertheless, both dielectrophoresis and acoustic 3D trap allow in-situ single-cell handling. However, single-cell manipulation is a complex task and needs to overcome various challenges (such as increasing throughput and experimental repeatability for robust design flexibility) in control over cells and tools that can allow handling of many isolated cells simultaneously with high specificity. Throughout this article, we have discussed lab-on-chip, microfluidic Bio-MEMS, and µTAS platform for cellular characterization and analysis. We interpret that the scope lies in the development of different tools and their applications. There is a wide gap in the availability of the current technology and requirement of actual tools. For example, antibody-antigen reactions are currently operated at a bulk level, which may conceal the low-frequency information of migrating cancer cells. Another problem is the incubation time required for these tests. Diagnostic tests have other limitations as well, which include, among others, the availability of appropriate reagents at all times. Hence, the applications of lab-on-a-chip technologies have garnered interest in overcoming these problems for precise diagnosis, and have been found to be the origin of most applications for single-cell technology development. This area of research needs more attention in the future. Cell isolation, sorting, detection and analysis hold great promise for disease diagnosis. Blood is a medium of transport for various cells throughout the body. Thus, liquid biopsy can potentially provide relevant information for any disease. A few of the major challenges faced in this field are low targeted cell concentration, specific antibodies for capturing targeted cells, and unwanted cellular interference. Another important obstruction is lack of spatio-temporal context due to which downstream analysis becomes impossible [100]. There is huge diversity in omics analysis, making it more challenging for direct detection or expression of targeted genetic materials. Further, omics analysis requires high sample purity in adequate quantities. Among omics technologies, proteomics proves to be much more complex than genetic material analysis, mainly due to the lack of protein amplification techniques and lack of highly specific affinity probes [249]. The entire process is a combination of various steps, making it an interdisciplinary task. The techniques that we discussed in the article pave the way for a solution to some of the above challenges. Cells manipulation and drug delivery through transient hydrophilic membrane pores involve sophisticated methods. In the future, modification and control of cell environment should be enabled such that cells can behave normally. Thus, regulating the cell microenvironment along with cell treatment can be the appropriate perspective towards treatment. It is necessary to understand the cause and changes in cell microenvironments, which is different for different cell individuals. Hence, the same medications do not have the same effect on different individuals. Thus, the requirement of individual medication arises. Endoscopy tools can be modified and incorporated with the lab-on-chip Int. J. Mol. Sci. 2018, 19, 3143 33 of 47 device, to analyze the cancer microenvironment and compare with the non-cancer environment of the same individual to understand the imbalance. Since a chemical imbalance is only a potent form of energy imbalance within the body, manipulation tools are necessary at every stage of this process. The packaging of required setup into a compact form is essential for an effective and efficient manipulation. Figure 33 shows the schematic representation of applications of single-cell technologies. Multiplexed point of care (xPOCT) is an emerging field for personalized medicine. Precise and early diagnosis is the critical step towards treatment of any disease. Prescription of treatment for diseases based on single biomarkers obtained through screening can be ambiguous. Hence, we need techniques that are highly sensitive in detecting multiple biomarkers for a diseased condition. Towards this purpose, Bio-MEMS devices are already available and show a lot of potential for advancement. The advantages of Bio-MEMS devices include easy-to-use non-intervention-based systems, low sample requirement, and highly accurate results, compared to bulk pathological techniques. Thus, incorporating single cell technology chips is a prospective advancement in xPOCT [250]. SCT also has wide applications for cellular therapeutic development. We introduced the most popular and upcoming physical methods, such as electroporation, optoporation, and mechanoporation, for cellular therapy and analysis. While other techniques (such as sonoporation [251], gene gun [252] and magnetoporation are also available [253]), they suffer from a lack of control for single-cell transfection (with the exception of electroporation and photoporation). Another important tool for delivery is microinjection, which offers [254] low throughput, low repeatability, and lack of robustness. Electroporation has been used for creating transient cell membrane pores while electrophoresis enhances movement of nanoscale-sized charged particles into cells [255]. While constrictions-based devices are still novel in the field of mechanoporation, they show a lot of promise, owing to their high transfection rates and ability to successfully deliver tough cargo such as CRISPR-Cas9 complexes into cells [256]. These devices report a higher cell viability rate after treatment in comparison to other physical therapeutic methods such as electroporation. Furthermore, Ding et al. designed a device incorporating both electroporation and mechanoporation for nuclear transfection of DNA into mammalian cells, thus introducing a strong precedent for hybrid devices in the future [257]. We can expect to see more devices combining different transfection techniques in advantageous ways to achieve transfection of tough cargo like never before. Over the last two decades, techniques for single-cell imaging have shown tremendous development. Integrating it with various manipulation technologies can allow improvement of cellular diagnostic and therapeutic applications. In addition, real-time monitoring techniques have made imaging-based analysis convenient. SCT has contributed to the development of many potent tools to detect cancer cells, illuminate epigenetic variations, analyze, and personalize treatment, among others. Such information on epigenetic variations can provide important information on neuronal degenerative diseases, such as Alzheimer’s Disease, and Parkinson’s Disease. Many single cell technologies have been employed in cancer research in different capacities, such as the use of RNAseq in the study of hetereogeneity of tumors [258], enabling anti-cancer treatment resistance by using single cell detection [259], and the recent scTrio-seq technology introduced by Hou et al. [260]. Another important application in SCT is stem cell research, which opens new frontiers for organ transplantation. SCT in stem cell research has contributed to finding dormant neural cells, which undergo division in accidental situations [261]. To reach the ultimate goal of routine application for clinical settings, two key contests are important. Firstly, investigational costs and time consumption is comparatively high; these need to be abridged to a rational range. Furthermore, it is essential to establish several techniques to test samples, which is repetitive preparation to keep tissue samples in the clinic. This progress will support the clinical application of SCT, which has positive outlooks for cancer diagnostics and treatment for cancer patients. Int. J. Mol. Sci. 2018, 19, 3143 34 of 47 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 34 of 46

FigureFigure 33. 33.Applications Applications of of single-cell single-cell technologies.technologies. Reprinted Reprinted with with permission permission from from [29,250]. [29,250 ].

7. Conclusions7. Conclusions InIn this this review review article, article, we we have have discusseddiscussed the dev developmentelopment of of SCT. SCT. The The entire entire article article gives gives a broad a broad perspectiveperspective to to the the reader reader about about the the steps steps involvedinvolved in the progress progress of of SCT SCT for for diseases diseases analysis. analysis. It also It also deliberatesdeliberates upon upon the the knowledge knowledge ofof currentcurrent SCTSCT progressprogress and and its its future future trends. trends. The The article article includes includes somesome of of the the recently recently developed developed SCT SCT toolstools involvedinvolved in disease diagnosis, diagnosis, analyzing analyzing it itin insingle-cell single-cell platformplatform and and also also treating treating it it using using novelnovel therapeutictherapeutic techniques for for cargo cargo delivery. delivery. The The diagnosis diagnosis mainlymainly highlights highlights the the manipulation, manipulation, detection,detection, sorting, and and isolation isolation of of single-cells single-cells or or diseased diseased cells cells fromfrom a bulka bulk of of heterogeneous heterogeneous cell cell types. types. Lab-on-chip is is a a well-suited well-suited technique technique that that integrates integrates multiple single-cell technologies based on downstream processing requirements. The combined use multiple single-cell technologies based on downstream processing requirements. The combined use of of microfluidics, laser optics, electrophoretic and electrochemical technology are discussed for single microfluidics, laser optics, electrophoretic and electrochemical technology are discussed for single cell cell diagnosis. Subsequently, we also discussed the devices that are useful for analyzing individual diagnosis. Subsequently, we also discussed the devices that are useful for analyzing individual cells cells to understand their physical and chemical interaction with the microenvironment. In addition, to understand their physical and chemical interaction with the microenvironment. In addition, we we demonstrated the application of SCT and other tools that have been developed to obtain demonstratedtherapeutics theand application drug delivery of SCT results and with other high tools cell that viability. have been Thisdeveloped review summarizes to obtain therapeuticsthe recent andtrends drug of delivery single-cell results technologies with high applied cell viability. for single-cell This reviewmanipulation, summarizes diagnosis, the recent therapeutics, trends of single-cellanalysis, technologiesdrug delivery applied and their for fu single-cellture prospects manipulation, and limitations. diagnosis, therapeutics, analysis, drug delivery and their future prospects and limitations. Author Contributions: P.S., L.M., A.K., K.D. and A.M. have. contributed to the content of the paper. A.N.P. has Authorcorrected Contributions: the entire manuscriptP.S., L.M., for A.K., grammar K.D. and and A.M. language. have. H.-Y.C. contributed has given to the biological content suggestions of the paper. to A.N.P.the hasmanuscript. corrected theF.-G.T. entire and manuscript M.N. have for given grammar technical and sugge language.stions for H.-Y.C. the manuscript. has given biological T.S.S. has suggestionstechnically and to the manuscript. F.-G.T. and M.N. have given technical suggestions for the manuscript. T.S.S. has technically and grammatically edited the manuscript. grammatically edited the manuscript. Int. J. Mol. Sci. 2018, 19, 3143 35 of 47

Acknowledgments: The authors greatly appreciate the financial support from Science and Engineering Research Board (SERB) under the grant number ECR/2016/001945, Department of Science and Technology (DST), Government of India and Wellcome Trust/DBT India Alliance Fellowship under grant number IA/E/16/1/503062. We acknowledge all authors and publishers from whom we got the copyright permissions. We acknowledge Srabani Kar for her help in preparing the graphical abstract. Conflicts of Interest: The authors declared no conflict of interest.

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