arXiv:1502.04991v1 [q-bio.TO] 17 Feb 2015 ilclnz h tutr.A h n fthe of end the At which structure. cells the living colonize with scaf- populated will The then first is bio-materials. fold is certain geometry using the a organ/tissue printed the printing, As of scaffold-based skeleton printing. in based suggests, scaffold-free scaffold names groups: and main printing two as egorized [5]. [4], printing [3], the [2], during process ecountered are viscos- that fluid etc.) pressure, ity stresses(friction, live to of on cells sensitivity the focused accomodate have to researchers techqniues the decades, Over to two tissue. adpated last human been material. complex now has manufacture of technique deposition same The layered with through as manufactured be ease such could tooling molds man- mutli-part expensive be Com- not without could manufacturing. ufactured previously that of shapes world plex the changed I • CONFERENCE BIOMEDICAL LAKES GREAT • I 1 rnigpoesfrbopitr a ecat- be can bio-printers for process Printing ehdb hre .Hl n18 [1], 1986 in Hull W. Charles by method NTRODUCTION tuiest fWisconsin-Milwaukee. of university at .Bksiea saPDsueti ehnclEngineering Mechanical [email protected] in Wisconsin-Milwaukee. E-mail: of student University PhD of a Department is Bakhshinejad A. ..DSuai soit rfso nmcaia engineer mechanical in Professor Associate is D’Souza R.M. Keywords viabilit t cell of and each density in cell methods speed, printing state-of-the-art resolution, review we paper this In e eod.Teltrhv oe pta eouin (100s resolutions spatial lower have later The second). per sitdadlsrfe.Tefre aevr n pta res spatial fine very t have human former The artificial free. fabricate laser to and assisted printing 3-D research on Recently, based tissue. methods human artificial manufacturing h ierqie o ua rasgetyicesstetim the increases greatly the trials where human environments for required time the Abstract NTRODUCTION re oprsnBtenAvailable Between Comparison Brief A Tesact fogn o rnpathsldt ag waiti large to led has transplant for organs of scarcity —The Bopitn,Tsu Engineering. Tissue —Bio-printing, ftefis Dprinting 3D first the of in − vivo l ahhnjdRsa D’Souza M Bakhshinejad,Roshan Ali i-rnigMethods Bio-printing odtoscnb lsl elctd ohteepolm c problems these Both replicated. closely be can conditions y. ing r ntsu niern aedvlpdtsu generation tissue developed have engineering tissue in ers ✦ ltos(0 of (10s olutions of omre.Du opne r erhn o alternative for searching are companies Drug market. to e eecassadpoieacmaio ae nreported on based comparison a provide and classes hese su.Body hs ehd ol ecasfida laser- as classified be could methods these Broadly, issue. vial rnigmtosada h n a end the of at and couple address methods briefly last will printing we available the review this during In decades. developed have been that techniques Each multiple (LfBP). contains bio-printing category free into Laser bio-printing categorized and assisted (LaBP) Laser be classes: can general two that bio-printers use [9]. cells from to away nutrients matter of waste diffusion and transport where cells handle systems bio- cannot systems in death alone in vascular cell challenge of to leads big Lack processes. a printing is vasculature func- of tional manufacture The [8]. gen- structure[7], automatically erate which signaling fusion cell cell through and occur sorting cell pro- as cell such Natural cesses substrate. a onto printed rectly other [6]. 1b) the (Figure layers and scaffolds on prefabricated printed 1a), be of will (Figure cells when cells is approach time living same the the when at out as is printed be approach will scaffold first the into approaches: categorized main be two Scaffold- further can cells. diffusion methods living ofbased the inward diffusion from enable outward materials and waste to oxygen de- way nutrients, is of a porosity in material signed Scaffold into systems. resolves the scaffold the process, colonization µ )btaevr at(pto (up fast very are but m) ohsafl-aeo cfodfe methods scaffold-free or scaffold-base Both di- are cells living printing, scaffold-free In glsso eysc ains ndu development, drug In patients. sick very of lists ng µ )btsfe rmso pe ( speed slow from suffer but m) 5 × 10 udb drse by addressed be ould 3 rp e second). per drops < 10 2 drops 1 GREAT LAKES BIOMEDICAL CONFERENCE 2

(a) (b)

Fig. 1: Schematic representation of (a) scaffold-base printing when bio-materials and cells will be printed at the same time (b) cell printing where cells will be place on prefabricated scaffolds [6].

comparison between different methods will be maintained in the range of 30−100µm. The lack presented. of a fluid orifice and reservoir in these designs means that issues such as print head clogging 2 LASER ASSISTED BIO-PRINTERS do not exist. However, the low printing speed 102 (LABP) (as low as drops/s) makes this process unsuitable for organ printing where the amount Laser assisted bio-printers (LaBP) were the first of material that must be deposited makes the type of bio-printers introduced by Odde and turn around time prohibitive. Renn [10] under name of ”Laser-Guided Direct . Writing” (LG DB). The printing setup included a laser beam, a focusing system and a substrate. The laser beam with the focusing system is 3 LASER FREE BIO-PRINTERS (LFBP) used to create a ”light trap” which is used to Laser free bio-printers were inspired by inkjet guide livig cells on the substracte to desired desktop printers. Inkjet printers typically con- locations. The long and direct laser light contact sists of a reservoir tank, micrometer size ori- with cells caused low cell survival rates[10]. fice, and a print head that can be actuated Subsequently, the principle of laser-induced either thermally, piezoelectrically (Figure 2) or forward transfer (LIFT), initially developed for by solenoid valves (Figure 3) [15], [14] . In inkjet metals, has been adpated to bio-printing and is printers, droplets are generated only when re- currently the main method for LaBP [11]. In a quired. A pressure pulse is generated in the typical LaBP, which is printing based on LIFT tank which force the bio-ink to go through method, (figure 2 ), focused laser pulses cause the orifice which leads to the printer head. local vaporization of an energy absorbing layer For printers that are equipped with a ther- (typically gold or titanium). This causes gen- mal print head, a micro-heater element va- eration of a droplet based on the energy of the porizes small pockets of the bio-ink to pro- pulse and the duration of the pulse. The size of duce vapor bubbles. Generation and collapse the droplet can be adjusted to meet the require- of these bubbles cause discontinuity in the bio- ments of the process [2], [12]. The generated ink stream. In piezoelectric models, the print droplets will be received by a substrate facing head is equipped with a piezoelectric actuator to the bio-ink [13], [14]. With this configuration, which deforms with a controlled frequency to the survival rate of cells was improved drasti- make discontinuity in the fluid stream [5]. In cally (up to 95% cell viability) since the laser the solenoid valve print head discontinuity in doesn’t have direct contact with live cells [11]. bio-fluid steam is be caused by a valve that can Simultaneously, the spatial resolution could be be turned on and off [15]. In the all mentioned GREAT LAKES BIOMEDICAL CONFERENCE 3

Fig. 2: Bio-printers with different printing approach. [14]

the robotic dispensing method has the lowest cell viability rates.(40 − 80%). Other LfBP are reported higher viability (> 85%) [16], [13], [14].

4 DISCUSSION

There are several different configurations avail- able for bio-printers. A end user has to choose and appropriate technique based on his or her requirements. Table 1 illustrates a comparison Fig. 3: Dispensing system for printer heads between different bio-printing techniques that equipped with solenoid valves [15] are currently available. Laser guided direct write (LG DW) printer have the best spatial resolution and the lowest viability. LIFT design was a big step toward configurations, the print head is connected to a improvement of cells viability for laser assisted robotic base controlled by software. Therefore printers. Modification of LIFT (as presented the print head can be accurately positioned in the table 1 as modified LIFT) added an over a substrate and the bio-ink droplet can be energy observing layer to the print head. This released at the desired location. change had a huge positive impact on cell In contrast with inkjet printers (generating viability. Laser free printing methods sacrifice droplets on demand) [3], in robotic dispens- cell density and spatial resolution to achieve ing, there is no droplet generation process. In- higher throughput. Print jobs that have intricate stead, the print head will produce a continuous features are therefore more suitable for LaBP stream of the bio-fluid and place it on the class of bio-printers. Print jobs with large tissue substrate. A controlled pressure system will masses are more suited for LfBP methods. forces the bio-ink to go through the nozzle. Depending on the viscosity of the fluid which requires different levels of the control, different pressure control systems can be utilized (Figure ACKNOWLEDGMENTS 2). The robotic dispensing method has the high- This work was partially supported by Na- est printing speed (10 − 50µm/s) as well as tional Institutes of Health, NIH/IU Subcontract high cell density. Among the LfBP systems, 2R01GM077138-04A1. GREAT LAKES BIOMEDICAL CONFERENCE 4

Printer type Resolution (µm) Print speed Cell viability(%) Cell densities References LaBP − LG DW 10-30 Continuous (9 × 10 8mL/s) Not Available as percentage 108cells/ml [10], [17], [18] Modified LIFT 30-100 102 drops/s 95-100 108cells/ml [11], [12], [17], [18] LfBP Thermal > 300 5 × 103 drops/s 75-90 < 106cells/ml [5], [17], [18] Piezoelectric - 1 × 104 drops/s (> 85%) < 106cells/ml [5], [17], [18] Solenoid valve - 6500 drops/s 85-99 5 × 105cells/ml [15], [17], [18] Robotic dispensing 5µm to millimeters 10 − 50µm/s 40-80 % cell spheroids [19], [20], [17], [18] TABLE 1: A brief comparison between bio-printers.

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