Droplet Microfluidics for the Ex Vivo Expansion of Human Primary

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Article Droplet Microﬂuidics for the ex Vivo Expansion of Human Primary Multiple Myeloma Cells

Pilar Carreras 1,2,*, Iciar Gonzalez 1 , Miguel Gallardo 2,3, Alejandra Ortiz-Ruiz 2,3 and Joaquin Martinez-Lopez 2,3,4

1 CSIC, Spanish National Research Council, 28006 Madrid, Spain; [email protected] 2 Hospital 12 Octubre, Hematology Department, Research institute i+12, 28040 Madrid, Spain; [email protected] (M.G.); [email protected] (A.O.-R.); [email protected] (J.M.-L.) 3 CNIO, Spanish national cancer research Centre, Hematological malignancies research unit, 28029 Madrid, Spain 4 UCM, Complutense University Madrid, Medical faculty, 28040 Madrid, Spain * Correspondence: [email protected]

Received: 21 January 2020; Accepted: 25 February 2020; Published: 29 February 2020

Abstract: We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device We demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double layered bead composed by an inner layer of alginate and an outer layer of puramatrix constructed using a soft technology without the use of any external force. In this work, we use this micro manipulation technique to build a 3D scaffold as a biomimetic model to recapitulate the niche of patient-derived multiple myeloma cells (MM cell) using a multilayered 3D tissue scaffold constructed in a microfluidic device and cultured in 10% FBS culture medium. In the current study, we included the use of this biomimetic model comprising supporting human Mesenchymal stem cells to show the mid-term survival of MM cells in the proposed structures. We found that the generated microniches were suitable for the maintenance of MM cells with and without supporting cells. Additionally, cultured MM cells in droplets were exposed to both Bortezomib and Lenalidomide to test their toxicity in the cultured patient derived cells. Results indicate that the maintained MM cells were consistently responding to the applied medication, opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening.

Keywords: microdroplets; microﬂuidics; stem cell encapsulation

1. Introduction Previous studies have been reported for the recapitulation of the stem cell microniche by droplet microfluidics [1]. Ex vivo maintenance and expansion of primary human multiple myeloma (MM) cells have presented several difficulties [2] due to the lack of an in vitro method able to reproduce the complex bone marrow niche microenvironment. Some tissue models have been reported for the ex vivo culture of primary MM cells [3–5]. Whereas these approaches have been successful in achieving MM cells maintenance for a time span of several weeks, the process to reach these results has been quite complex as several supporting cells and/or complex perfusion systems were requested [6]. Microfluidics have recently arisen as a promising technology for 3D cell culture as it provides the possibility of generating biomimetic structures in which native stem cell biological processes can be recapitulated [7]. Three-dimensional cell culture systems have included many scaffold-free [8,9] and scaffold supported systems [10,11]. Stem cell maintenance studies in these structures have also been performed in suspension or in a spinner flask,

Micromachines 2020, 11, 261; doi:10.3390/mi11030261 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 261 2 of 9 resulting in a large heterogeneity in spheroid size [12], and in hanging-drop techniques that provide some control over the spheroid size but which lack aggregated generation control [13,14]. Whereas several methods able to form an aggregation of cell structures have been reported, most of them showed either a lack of stability on the template [15], or control on the geometry of the cell constructs [16]. Previous studies have reported the generation of a MM niche by microfluidics techniques [17]. However, these techniques were complex to perform and most of the time soluble additive factors were required for its growth and maintenance [18,19]. Hydrogels have been proved to be successfully used for 3D cell culture purposes [20,21]. While alginate is the most well-known hydrogel in cell culture studies, there is a growing interest in microengineering materials for improving the extracellular matrix recapitulation [22]. Recently we reported a method in which a double layered hydrogel bead able to accommodate adult stem cells where controlled generation of the structure geometry, composition and cell distribution was achieved [1]. This technology has been improved and accommodated to culture MM stem cells as part of the wide field of applicability that the method can offer for life science studies. In the presented droplet-based microdevice, hydrogel droplets are produced by hydrodynamic focusing techniques and gelled by the circulation of the droplet between two laminar flows, one containing the cross-linking agent. This generated droplet is coated by another synchronized hydrogel bead as previously reported, generating on demand three-dimensional niches for stem cell allocation by synchronizing droplet production rates. The combination of these processes of droplet generation and passive mixing without the use of external forces makes this technology suitable for the encapsulation of stem cells [1]. The presented method permits the rapid extraction of the generated cellular constructs for immediate washing and culture, limiting the time exposure of the cells to the cross-linking agent. This feature allows the successful culture of cells like MM cells which usually present difficulties to be maintained ex vivo. This new technology does not require a complex setup or affective growth factor and represents a promising technique able to provide optimal requirements for 3D multiple myeloma (MM) stem cell culture for personalized medicine applications and ex vivo drug testing.

2. Materials and Methods

2.1. Materials/Chemicals Mineral oil (Sigma Aldrich, St. Louis, MO, USA) was used as the main oil carrier. Sudan red dye (Sigma Aldrich) was used to color the oil containing the cross-linking agent and visualize the interface between the two main carrier oil ﬂows. Alginic acid sodium salt powder (Sigma Aldrich) was used to prepare the alginate solutions. The aqueous phase was composed of deionized water unless otherwise stated. Puramatrix 0, 3% (Becton Dickinson) solution was prepared at the corresponding concentrations by sonication of the gel for 30 min at 30 ◦C. Acetic acid (Sigma Aldrich) was used as the cross-linking agent. Nano-calcium carbonate powder was purchased from Skyspring Nanomaterials (Houston, TX, USA) and used following the vendor instructions (https://www.ssnano.com).

2.2. Cell Assays Phosphate Buffered Saline PBS was acquired from Cultek. Calcein-AM (0.1 ug/uL) and DAPI (40, 6-diamidino-2-phenylindole), 10 mg (200ug/mL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CD38 FITC (fluorescein isothiocyanate) was purchased from Becton Dickinson, US. Phosphate Buffered Saline PBS was acquired from Cultek (Madrid, Spain). Patient derived MM cells were collected and inmunomagnetically purified with written consent in accordance with the Institutional Review Board of Hospital 12 October Madrid and in accordance with the Declaration of Helsinki. Micromachines 2020, 11, x 3 of 9

Patient: MM IgG Kappa IIIA ISS I patient treated with VTDx6 + TASPE regimen, Rt +KRD after 1st relapse, PomCyDex + RT after 2nd relapse and Selinexor+Bortezomib+Dexametasone regiment after 3rd relapse, in partial response at the moment of the sample. Human Mesenchymal stem cells were purchased from DSMZ. The cell line was cultured in DMEM (Biowest, France) supplemented with 10% fetal bovine serum. Cells were cultured and used from passages 2–6 in a 37 °C humidified incubator with 5% CO2. Trypsin-EDTA (0.25%), phenol red was acquired from Thermo Fisher Scientific (Waltham, MA, USA).

2.3. Microfabrication Micromachines 2020, 11, 261 3 of 9 The microfluidic devices were designed using Solidworks CAD (Dassault Systèmes, Vélizy- Villacoublay, France) software. A computer numerical control (CNC) program was then created usingPatient: Mastercam MM IgG(CNC Kappa Software IIIA ISSInc., I Tolland, patient treated CT, USA with), according VTDx6 + toTASPE the microfluidic regimen, Rt circuit+KRD design after 1stand relapse, dimensions. PomCyDex The outline+ RT after of the 2nd device relapse was and extracted Selinexor from+Bortezomib a PMMA +(polymethylmethacrylate)Dexametasone regiment afterplastic 3rd sheet relapse, (MacMaster in partial-Carr response, Elmhurst, at the IL, moment USA) ofusing the a sample. Laser engraver (VLS 6.30, Universal Laser Systems,Human Scot Mesenchymaltsdale, AZ, USA stem). cellsThe wereplastic purchased cut out was from then DSMZ. taken The tocell a CNC line wasmachine cultured (HAAS in DMEM Super (Biowest,Minimill, France)Oxnard, supplemented CA, USA), where with the 10% microfluidic fetal bovine circuit serum. was Cells created were based cultured on andthe usedMastercam from passagesprogram. 2–6 Milling in a 37 conditions◦C humidified: milling incubator direction, with spindle 5% CO2. speed Trypsin-EDTA and feed rate(0.25%), were phenol optimized red was for acquiredsurface quality. from Thermo Finally, Fisher fluidic Scientific ports were (Waltham, created MA,using USA). a drill press (Ellis). The PMMA device was sealed with a self-adhesive pouch (Opko diagnosis, Miami, FL, USA). 2.3.Main Microfabrication channel dimensions are displayed in Figure 1 The device was connected to open barrels of 10 mL disposableThe microfluidic plastic syringes devices (BD were biosciences) designed via usingsilicone Solidworks tubing (ID 1.1 CAD mm O.D. (Dassault 2.16 mm) Syst (Fisherèmes, Vscientific).élizy-Villacoublay, The driving France) force software. used Afor computer the experiments numerical was control gravity, (CNC) so program liquid was pressure then created on the usingmicrodevice Mastercam was (CNCcontrolled Software by the Inc., heights Tolland, of CT,the USA),open accordingsyringes. Pieces to the microfluidicof polytetrafluoroethylene circuit design and(PTFE) dimensions. tubing (OD The 2 mm) outline (Cole of theParmer device, Vernon was extracted Hills, IL, fromUSA) a were PMMA used (polymethylmethacrylate) to interface the reservoirs plasticto the chip. sheet (MacMaster-Carr, Elmhurst, IL, USA) using a Laser engraver (VLS 6.30, Universal Laser Systems, Scottsdale, AZ, USA). The plastic cut out was then taken to a CNC machine (HAAS Super Minimill,2.4. Microscopy Oxnard, CA, USA), where the microfluidic circuit was created based on the Mastercam program.Fluorescence Milling conditions: images were milling acquired direction, using spindle inverte speedd laser and scanning feed rate confocal were optimizedmicroscopy for (LSM510 surface quality.META, Finally,Axiovert fluidic RT 200M, ports Zeiss, were createdOberkochen, using Germany) a drill press equipped (Ellis). with an environmental chamber maintainingThe PMMA the cells device under was physiological sealed with acondition self-adhesives, using pouch the laser (Opko lines diagnosis, 405 nm and Miami, 488 nm. FL, USA).For all Mainother channelexperiments dimensions, a Motic areStereoscope displayed (Motic in Figure, Hong1 The Kong device, China was) and connected Moticam to2.0 opensoftware barrels (Motic) of 10were mL used. disposable plastic syringes (BD biosciences) via silicone tubing (ID 1.1 mm O.D. 2.16 mm) (Fisher scientific). The driving force used for the experiments was gravity, so liquid pressure on the microdevice2.5. Research wasMethodology controlled by the heights of the open syringes. Pieces of polytetrafluoroethylene (PTFE) tubing (OD 2 mm) (Cole Parmer, Vernon Hills, IL, USA) were used to interface the reservoirs to The schematic of the device used to encapsulate MM cells is depicted in Figure 1. the chip.

Figure 1. (a) Schematic of the double layered hydrogel bead generator. (b) Autocad layout of the microﬂuidic device. (c) MM (multiple myeloma) microniche models used. Top: double layered bead Figure 1. (a) Schematic of the double layered hydrogel bead generator. (b) Autocad layout of the containing human multiple myeloma cells in the outer layer and human Mesenchymal stem cells in the microfluidic device. (c) MM (multiple myeloma) microniche models used. Top: double layered bead inner core. Bottom: double layered bead containing human MM cells in the outer layer and an empty alginatecontaining core. human multiple myeloma cells in the outer layer and human Mesenchymal stem cells in

2.4. Microscopy Fluorescence images were acquired using inverted laser scanning confocal microscopy (LSM510 META, Axiovert RT 200M, Zeiss, Oberkochen, Germany) equipped with an environmental chamber maintaining the cells under physiological conditions, using the laser lines 405 nm and 488 nm. For all other experiments, a Motic Stereoscope (Motic, Hong Kong, China) and Moticam 2.0 software (Motic) were used. Micromachines 2020 11 Micromachines 2020, , 11, 261, x 44 of of 9 9

the inner core. Bottom: double layered bead containing human MM cells in the outer layer and an 2.5. Research Methodology empty alginate core. The schematic of the device used to encapsulate MM cells is depicted in Figure1. First,First, alginate alginate droplets droplets containing containing calcium calcium carbonate carbonate nanopowder nanopowder are generated are by hydrodynamic generated by focusinghydrodynamic using gravity focusing to drive using pressure. gravity to The drive droplets pressure. then travel The droplets through a then meandering travel through channel a containingmeandering two channel laminar containing oil flows. By two the laminar addition oil of aceticflows. acid By tothe the addition second laminarof acetic oil acid flow to continuous the second phase,laminar calcium oil flow ions continuous are released phase, after calcium droplet formationions are released by the diafterffusion droplet of the formation acetic acid by across the diffusion the oil interface.of the acetic The acid so generated across the droplets oil interface. are then The passed so generated by a second droplets inlet are were then a stream passed of by second a second sample inlet layerwere alginate a stream droplets of second are sample generated layer in alginate a synchronous droplets way. are generated in a synchronous way. Hence,Hence, when when the the inner inner core core droplet droplet reaches reaches the the area area of of the the second second droplet droplet production, production, the the two two dropletsdroplets (one (one gelated gelated and and another another ungelated) ungelated) passively passively mix mix along along the the meander, meander, generating generating a a double double layeredlayered hydrogel hydrogel bead. bead. This This process process was was described described in in detail detail in in the the previously previously reported reported work work [1 [].1]. FurtherFurther characterization characterization of of the the mixing mixing e ffiefficiencyciency of of the the reported reported device device is is depicted depicted in in Figure Figure2. 2.

Figure 2. Characterization for the double layered device operational conditions using droplet different combinationsFigure 2. Characterization of Puramatrix(0.3%) for the and double alginate layered left: (1.5%) device and operational right:(1%) containing conditions calcium using droplet nano powderdifferent (1 combinations wt%). of Puramatrix(0.3%) and alginate left: (1.5%) and right:(1%) containing calcium nano powder (1 wt%). 3. Results 3. ResultsSamples were generated by premixing aqueous samples containing alginate (1.5 wt%) and Calcium carbonate nanopowder (1 wt%) and were prepared as sample 1 for the generation of the inner core. Samples were generated by premixing aqueous samples containing alginate (1.5 wt%) and Puramatrix (0.3% v/v) was prepared following the vendor instructions for the generation of the outer Calcium carbonate nanopowder (1 wt%) and were prepared as sample 1 for the generation of the layer. The second oil was prepared by adding glacial acetic acid to a pre-dyed Sudan red mineral oil inner core. Puramatrix (0.3% v/v) was prepared following the vendor instructions for the generation (0.5% v/v). of the outer layer. The second oil was prepared by adding glacial acetic acid to a pre-dyed Sudan red Adjustments of the samples’ flow rates were performed so that the inner core produced was mineral oil (0.5% v/v). synchronized with the secondhand allowing its collection (Figure2). Once the experimental flow rates Adjustments of the samples’ flow rates were performed so that the inner core produced was were set up for the synchronization of the inner core droplet and outer layer droplet, characterization synchronized with the secondhand allowing its collection (Figure 2). Once the experimental flow studies were run by varying the height of the oil 2 (oil containing the gelating agent) and using alginate rates were set up for the synchronization of the inner core droplet and outer layer droplet, as a scaffold in the inner core and puramatrix in two different concentrations for the outer layer. characterization studies were run by varying the height of the oil 2 ( oil containing the gelating agent) These measurements confirmed the already demonstrated capacity of the device to synchronize the and using alginate as a scaffold in the inner core and puramatrix in two different concentrations for hydrogel beads for the experimental conditions to study with the MM cells. the outer layer. These measurements confirmed the already demonstrated capacity of the device to synchronizMM cellse the were hydrogel purified beads from for a specific the experimental patient. The conditions suitability to of study the presented with the MM method cells for. MM cell encapsulationMM cells were was purified demonstrated from a specific by accommodating patient. The suitability primary patient of the presented derived human method Multiple for MM Myelomacell encapsulation cells in the was outer demonstrated layer. Three dibyff erentaccommodating conditions wereprimary set inpatient the same derived experiment. human TheMultiple first setMyeloma of experiments cells in the included outer layer the. generationThree different of a conditions mixed droplet were containingset in the same only experiment. human MM The cells. first Aset second of experi setments of collected included beads the wasgeneration planned of soa mixed that the droplet outer containing layer contained only human MM cells MM and cells the. A innersecond layer set consistedof collected of emptybeads alginate.was planned A third so th setat ofth experimentse outer layer wascontained prepared MM so cell thats and human the MMinner cellslayer were consist locateded of in empty the outer alginate. layer A and third supporting set of exper humaniments Mesenchymal was prepared stem so that cells human (hMSCs) MM were cells encapsulatedwere located into in the middle outer layer layer, and as these supporting cells are human naturally Mesenchymal residing on the stem MM cells microniche. (hMSCs) were encapsulated into the middle layer, as these cells are naturally residing on the MM microniche. MicromachinesMicromachines2020 2020,,11 11,, 261 x 55 ofof 99

Prior to resuspension in PBS and mixture in premixed alginate emulsification, human Prior to resuspension in PBS and mixture in premixed alginate emulsiﬁcation, human Mesenchymal Mesenchymal stem cells were routinely cultured and trypsinized. We used 5 × 106 cells/mL for stem cells were routinely cultured and trypsinized. We used 5 106 cells/mL for human MM and human MM and 0.7 × 106 cells/mL for hMSC. × 0.7 106 cells/mL for hMSC. × The generated cell laden structures were collected and washed to remove residual acid. FITC The generated cell laden structures were collected and washed to remove residual acid. FITC CD38 which fluoresces green upon the reaction of intracellular esterase and stains live cells, and DAPI CD38 which ﬂuoresces green upon the reaction of intracellular esterase and stains live cells, and DAPI 0.1 M which binds to the DNA of dead membrane compromised cells were employed to visualize the 0.1 M which binds to the DNA of dead membrane compromised cells were employed to visualize the distribution of human MM alive and dead cells right after encapsulation. distribution of human MM alive and dead cells right after encapsulation. Figure 3 shows the results of the collecting of these three sets of beads after 24, 48, 72 h and one Figure3 shows the results of the collecting of these three sets of beads after 24, 48, 72 h and one week of culture in 10% FBS (Fetal Bovine Serum) DMEM. Collected beads were imaged under week of culture in 10% FBS (Fetal Bovine Serum) DMEM. Collected beads were imaged under confocal confocal microscope; images were taken at the middle plane of the bead and then three more planes microscope; images were taken at the middle plane of the bead and then three more planes to the to the bottom along the z axis in order to visualize the maximum number of encapsulated cells. bottom along the z axis in order to visualize the maximum number of encapsulated cells.

FigureFigure 3.3. Collected beads after ( a)) 24 24 h h,, ( (bb)) 48 48 h h,, ( (cc)) 72 72 h h,, and and (d (d) )one one week. week. For For each each case case from from top top to tobottom: bottom: mixed mixed bead bead with with MM MM cells, cells, double double layer layer bead bead with with an an outer outer layer layer of MM cells andand emptyempty innerinner alginatealginate corecore andand doubledouble layerlayer beadbead withwith anan outerouter layerlayer ofof MMMM cellscells andand innerinner layerlayer ofof humanhuman MesenchymalMesenchymal stem stem cells cells (hMSC) (hMSC cells.) cells. For For each each case case from from left left to to right: right: di ﬀdifferenterent Z planeZ plane cuts cuts selected selected to includeto include the the maximum maximum number number of positiveof positive cells. cells.

ImagesImages were were analyzed analyzed for for the the first first week week of of culture culture and and cells cells were were manually manually counted counted using using Image Image J software.J software. Cells Cells were were manually manually counted counted over over a a minimum minimum of of ten ten beads beads per per condition condition andand takingtaking intointo accountaccount cellscells imagedimaged atat leastleast inin fourfour planesplanes alongalong thethe zz axisaxis inin orderorder toto quantifyquantify thethe numbernumber ofof positivepositive FITCFITC CD38CD38 cellscells andand DAPI.DAPI. ItIt waswas observedobserved thatthat cellcell numbersnumbers werewere increasingincreasing significantlysignificantly duringduring thethe firstfirst 7272 h.h. AfterAfter aa weekweek cellcell numbersnumbers werewere slightlyslightly decreaseddecreased forfor thethe doubledouble layeredlayered beadsbeads andand maintainedmaintained forfor thethe mixedmixed beadbead case.case. ResultsResults areare compiledcompiled inin FigureFigure4 .4. Beads were maintained in culture for two weeks performing three media changes per week. After two weeks of ex vivo MM culture, the collected beads were incubated with Bortezomib and Lenalidomide for 24 h. The frontline therapy for multiple myeloma patients is the combination of three drugs, bortezomib, lenalidomide and dexametasone. Bortezomib is a proteasome inhibitor (binds to 26S catalytic subunit) with high efficacy against multiple myeloma plasma cells. The in vitro IC50 in MM Micromachines 2020, 11, x 6 of 9 cell lines varies in a range of 2–40 nM, that is the reason 8 nM and 16 nM concentrations were selected for the experiment. On the other hand, lenalidome is an IMID (Immunomodulatory imide) drug. Lenalidome has a plethora of different targets (e.g., CRBN) and effects (e.g., immunomodulation, anti-angiogenic, pro- apoptotic, anti-osteoclastogenic) and so it could be impacted in MM plasma and MM MSC cell. Due to its ample mechanism of action, it has a limited and lower effect in vitro compared with in vivo, with uM IC50 ranges (1–10 uM). Therefore, the first test was designed to test bortezomib (concentration 8 nm) and lenalidomide Micromachines(concentrations2020, 111 uM, 261 and 2 uM (Figure 5)). 6 of 9

Micromachines 2020, 11, x 6 of 9

cell lines varies in a range of 2–40 nM, that is the reason 8 nM and 16 nM concentrations were selected for the experiment. On the other hand, lenalidome is an IMID (Immunomodulatory imide) drug. Lenalidome has a plethora of different targets (e.g., CRBN) and effects (e.g., immunomodulation, anti-angiogenic, pro - apoptotic,Figure 4. antiNumber-osteoclastogenic) of FITC CD38 and+ and so DAPI it could cells be for impacted the studied in cases.MM plasma From left and to right:MM MSC counted cell. Due to itscellsFigure ample for 4. mixed Numbermechanism beads, of FITC double of action,CD38+ layered andit beadshas DAPI a withoutlimited cells for MSCsa ndthe lowerstudied and Double effect cases. layeredin From vitro beadsleft compared to withright: MSCs. counted with in vivo, withcells uM for IC mixed50 ranges beads, (1– double10 uM layered). beads without MSCs and Double layered beads with MSCs. Beads were maintained in culture for two weeks performing three media changes per week. Therefore, the first test was designed to test bortezomib (concentration 8 nm) and lenalidomide After two weeks of ex vivo MM culture, the collected beads were incubated with Bortezomib and (concentrations 1 uM and 2 uM (Figure 5)). Lenalidomide for 24 h. The frontline therapy for multiple myeloma patients is the combination of three drugs, bortezomib, lenalidomide and dexametasone. Bortezomib is a proteasome inhibitor (binds to 26S catalytic subunit) with high efficacy against multiple myeloma plasma cells. The in vitro IC50 in MM cell lines varies in a range of 2–40 nM, that is the reason 8 nM and 16 nM concentrations were selected for the experiment. On the other hand, lenalidome is an IMID (Immunomodulatory imide) drug. Lenalidome has a plethoraFigure of5. Confocal different microscopy targets (e.g., imaging CRBN) of 4 and different effects z planes (e.g., immunomodulation,for beads marked using anti-angiogenic, FITC CD38 pro-apoptotic,and DAPI. Z anti-osteoclastogenic) planes were chosen to image and so the it maximum could be number impacted of cells. in MM From plasma top to andbottom: MM control MSC cell. Due tobead, its amplebead incubated mechanism with of b action,ortezomib it has 8 nM a limited, bead andincubated lower with effect lenalidomiin vitro comparedde 1 uM and with beadin vivo, withincubated uM IC50 rangeswith lenalidomide (1–10 uM). 2 uM. Therefore,Figure 4. Number the first of test FITC was CD38+ designed and DAPI to test cells bortezomib for the studied (concentration cases. From 8 nm)left to and right: lenalidomide counted (concentrationsIt cells was for observed mixed 1 uM beads, and that 2double uM cells (Figure layered were5 )).beads respond withouting significantlyMSCs and Double to thelayered incubation beads with process MSCs. with bortezomib 8 nM and very slightly responding to the incubation with lenalidomide 1 uM and 2 uM. To assure this conclusion, experiments were repeated two days afterwards exposing the beads to a double concentration of bortezomib 16 nM, 8 nM and lenalidomide 2 uL. Frames extracted from the confocal imaging are displayed in Figure 6. Cells were manually counted over a minimum of ten beads per condition and taking into account cells imaged at least in four planes along the z axis in order to quantify the number of positive FITC CD38 cells and DAPI. Results are shown in Figure 7.

Figure 5. Confocal microscopy imaging of 4 diﬀerent z planes for beads marked using FITC CD38 and DAPI.Figure Z planes5. Confocal were chosenmicroscopy to image imaging the maximum of 4 different number z planes of cells. for From beads top marked to bottom: using control FITC bead, CD38 beadand incubatedDAPI. Z planes with bortezomibwere chosen 8 to nM, image bead the incubated maximum with number lenalidomide of cells. 1From uM andtop to bead bottom: incubated control withbead, lenalidomide bead incubated 2 uM. with bortezomib 8 nM, bead incubated with lenalidomide 1 uM and bead incubated with lenalidomide 2 uM. It was observed that cells were responding signiﬁcantly to the incubation process with bortezomib 8 nM andIt was very observed slightly responding that cells wereto the respond incubationing with significantly lenalidomide to the 1 uM incubation and 2 uM. process To assure with thisbortezomib conclusion, 8 nM experiments and very slightly were repeated responding two to days the incubation afterwards with exposing lenalidomide the beads 1 uM to aand double 2 uM. concentrationTo assure this of conclusion, bortezomib e 16xperiments nM, 8 nM were and lenalidomide repeated two 2 days uL. Frames afterwards extracted exposing from the the bead confocals to a imagingdouble areconcentration displayed inof Figurebortezomib6. 16 nM, 8 nM and lenalidomide 2 uL. Frames extracted from the confocalCells wereimaging manually are displayed counted in over Figure a minimum 6. of ten beads per condition and taking into account cells imagedCells were at least manually in four planes counted along over the a z minimum axis in order of ten to quantify beads per the condition number of and positive taking FITC into CD38account cells cells and imaged DAPI. Resultsat least arein four shown planes in Figure along 7the. z axis in order to quantify the number of positive FITC CD38 cells and DAPI. Results are shown in Figure 7. MicromachinesMicromachines2020 2020, ,11 11,, 261 x 77 ofof 99 Micromachines 2020, 11, x 7 of 9

Figure 6. Confocal microscopy imaging of 4 different z planes for beads marked using FITC CD38 FigureFigure 6. Confocal 6. Confocal microscopy microscopy imaging imaging of 4of di 4ﬀ erentdifferent z planes z planes for beadsfor beads marked marked using using FITC FITC CD38 CD38 (green) and DAPI (blue). Z planes were chosen to image the maximum number of cells. From top to (green)(green and) and DAPI DAPI (blue). (blue Z) planes. Z planes were were chosen chosen to image to image the maximumthe maximum number number of cells. of cells. From From top totop to bottom: control bead, bead incubated with bortezomib 16 nM, bead incubated with bortezomib 8 nM bottom:bottom: control control bead, bead, bead bead incubated incubated with with bortezomib bortezomib 16 nM, 16 nM bead, bead incubated incubated with with bortezomib bortezom 8ib nM 8 nM and bead incubated with lenalidomide 2 uM. andand bead bead incubated incubated with with lenalidomide lenalidomide 2 uM. 2 uM.

Figure 7. Left: Number of FITC CD38 and DAPI cells per bead for the studied cases (control, bortezomib Figure 7. Left: Number of FITC CD38 and DAPI cells per bead for the studied cases (control, 16 nM,Figure 8 nM 7. andLeft lenalidomide: Number of 2 FITC uM). Right CD38: and Percentage DAPI cells of dead per cells bead for for control the stud beads,ied bortezomib cases (control, bortezomib 16 nM, 8 nM and lenalidomide 2 uM). Right: Percentage of dead cells for control beads, 1 nMbortezomib and 8 nM and16 nM lenalidomide, 8 nM and 2lenalidomide uM. 2 uM). Right: Percentage of dead cells for control beads, bortezomib 1 nM and 8 nM and lenalidomide 2 uM. bortezomib 1 nM and 8 nM and lenalidomide 2 uM. Results, for these specific patient cells, suggest as expected that the patient is responsive to Results, for these specific patient cells, suggest as expected that the patient is responsive to bortezomibResults, significantly for these whereas specific the patient effect cells,of lenalidomide suggest as is expected much lower. that Thethe patientresults displayed is responsive for to bortezomib significantly whereas the effect of lenalidomide is much lower. The results displayed for thesebortezomib specific patient significantly cells in whereas Figure7 showed the effect the of correct lenalidomide e fficacy is of much both lower anti-myeloma. The results drugs displayed against for these specific patient cells in figure 7 showed the correct efficacy of both anti-myeloma drugs against MMthese primary specific plasma patient cells cells (CD38 in figu+) inre the7 showed generated the correc microniche.t efficacy Thus, of both this anti experiment-myeloma is drugs a proof against of MM primary plasma cells (CD38+) in the generated microniche. Thus, this experiment is a proof of conceptMM thatprima thery generated plasma cells MM (CD38+) microniche in the is agenerated novel vehicle micro allowingniche. Thus, a successful this experiment culture of is primary a proof of concept that the generated MM microniche is a novel vehicle allowing a successful culture of primary MMconcept cells which that the are generated very diffi MMcult tomicro cultureniche in isother a novel conditions, vehicle allowing such as a 2D-culturesuccessful culture or co-cultures. of primary MM cells which are very difficult to culture in other conditions, such as 2D-culture or co-cultures. ThisMM could cells prove whi usefulch are asvery a drug difficult test platform.to culture in other conditions, such as 2D-culture or co-cultures. This could prove useful as a drug test platform. ThisThese could studies prove also useful indicate as a drug that thetest maintainedplatform. MM cells were consistently responding to the These studies also indicate that the maintained MM cells were consistently responding to the applied medication,These studies observing also indicate similar that eff ectsthe maintained of anti-myeloma MM cells againstwere consistently MM cell lines responding in standard to the applied medication, observing similar effects of anti-myeloma cells against MM cell lines in standard 2D-culture,applied openingmedication, a wide obse fieldrving of possibilitiessimilar effects to useof anti the- presentedmyeloma cells micro against device MM as an cell ex vivolines platform in standard 2D-culture, opening a wide field of possibilities to use the presented micro device as an ex vivo for drug2D-culture, screening. opening a wide field of possibilities to use the presented micro device as an ex vivo platform for drug screening. platform for drug screening. 4. Discussion and Conclusions 4. Discussion and Conclusions 4.The Discussion difficulties and to Con cultureclusions primary plasma cells of multiple myeloma patients suggest the urgency The difficulties to culture primary plasma cells of multiple myeloma patients suggest the to developThe a novel difficulties method to to culture study the primary MM PC plasma (multiple cells myeloma of multiple plasma myeloma cells) ex vivo. patients In the suggest current the urgency to develop a novel method to study the MM PC (multiple myeloma plasma cells) ex vivo. In manuscripturgency weto develop describe a a novel novel method protocol to to study successfully the MM harvest PC (multiple and amplify myeloma MM plasma PC in mid cells)/long-term ex vivo. In the current manuscript we describe a novel protocol to successfully harvest and amplify MM PC in cultures.the current The presented manuscript study we indicatesdescribe thata novel MM protocol cell viability to successfully is successfully harvest maintained and amplify over M atM least PC in mid/long-term cultures. The presented study indicates that MM cell viability is successfully mid/long-term cultures. The presented study indicates that MM cell viability is successfully Micromachines 2020, 11, 261 8 of 9 two weeks with the generation of the presented microniches by microfluidic techniques. The beads at week 2 showed still high viability which suggests its longer-term applicability. Patient derived cultured cells in droplets were also exposed to the patient’s treatment obtaining corresponding responses. These results suggest that these ex vivo maintained MM cells are functional and could be used as a therapeutic platform. The applications of this finding are remarkable. For instance, it could be used to amplify CD138+ cells in order to be applied for in vivo applications. However, the most prominent applicability probably is the use of therapeutic screening in this novel ex vivo platform with different regiment treatments (e.g., BTZ+Lenalidomide, secondary treatment lines, etc.) in order to determine to which treatment the cells are more sensitive In conclusion, we present a novel droplet-based culture system for the encapsulation and maintenance of human primary multiple myeloma (MM) stem cells. This novel technology is based on a passive mixing principle and a gelation system in which the use of a double laminar oil flow where only one contains the cross-linking agent allows the mild generating of double layered hydrogel beads containing stem cells on demand. The soft consecutive coating of the inner core with a second layer without exposing the encapsulated cells to external forces that might reduce their viability constitutes the main technology advance towards a platform for 3D stem cell encapsulation. Additionally, the suitability of the presented technology for encapsulation and maintenance of primary MM stem cells is demonstrated by using both only the studied MM stem cells and a combination of the MM stem cells and human Mesenchymal Stem cells (hMScs) in the inner layer as supporting cells. From the culture device perspective, our results demonstrate the possibility of replicating the multi-cellular MM niche in a simple and low-cost way and the possibility of a drug test directly over the cultured beads, characterizing the responses with confocal microscopy techniques. The presented device represents an efficient attempt to engineer stem cell niches ex vivo using a three-dimensional matrix, and a novel platform to study MM cell behavior in vitro under controlled conditions as well as a novel therapeutic platform to study drug effects directly on patient derived cultures. Therefore, the presented device is a valuable and unique tool towards personalized drug testing and understanding stem cell behavior in 3-dimensional environments.

Author Contributions: Conceptualization and methodology, P.C.; investigation, P.C.; imaging, P.C., A.O.-R.; analysis, P.C.; validation: P.C.; resources, P.C., I.G., M.G.; writing, review and editing P.C., M.G.; supervision, J.M.-L., I.G.; project administration, P.C., I.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by European Commission H2020 Marie Curie Research Grants Scheme MSCA-IF-GF (705163). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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