New Frontiers for Biofabrication and Bioreactor Design in ......typically by applying enabling technologies, including microfabricated moldsormicroﬂuidics’. Biofabrication: ‘The

Trends in Biotechnology

TIBTEC 1787 No. of Pages 17

Review

New Frontiers for Biofabrication and BioreactorDesign in MicrophysiologicalSystem Development

Jonathon Parrish,1,2 Khoon Lim,1,2,8,@ Boyang Zhang,3,9,@ Milica Radisic,4,5,6,7,10,@ andTim B.F. Woodfield1,2,11,*,@

HighlightsA range of advanced biofabrication tech-niques have been developed in recentyears to enhance our ability to positioncells and extracellular matrices in 3D,leading to the construction of increas-ingly complex tissues.

Complex organ-on-a-chip devices withmodular design and advanced fluid cir-culation control have been developed tomodel complex cellular communicationin tissues as well as interorgan

Microphysiological systems (MPSs) have been proposed as an improved tool torecreate the complex biological features of the native niche with the goal of improv-ing in vitro–in vivo extrapolation. In just over a decade, MPS technologies haveprogressed from single-tissue chips to multitissue plates with integrated pumpsfor perfusion. Concurrently, techniques for biofabrication of complex 3D constructsfor regenerative medicine and 3D in vitro models have evolved into a diverse tool-box for micrometer-scale deposition of cells and cell-laden bioinks. However, asthe complexity of biological models increases, experimental throughput is oftencompromised. This review discusses the existing disparity betweenMPS complex-ity and throughput, then examines anMPS-terminated biofabrication line to identifythe hurdles and potential approaches to overcoming this disparity.

interaction.

The need for more advanced tissuemodels with greater structural complex-ity and dynamic external stimulation mo-tivated the convergence of biofabricatedtissues and organ-on-a-chip devices inrecent years.

1Christchurch Regenerative Medicineand Tissue Engineering (CReaTE) Group,Department of Orthopaedic Surgery andMusculoskeletal Medicine, University ofOtago Christchurch, Christchurch,New Zealand2New Zealand Medical TechnologiesCentre of Research Excellence(MedTech CoRE), Auckland, NewZealand3Department of Chemical Engineering,McMaster University, Hamilton, ON,Canada4Department of Chemical Engineeringand Applied Chemistry, University ofToronto, Toronto, ON, Canada5Institute for Biomaterials and BiomedicalEngineering, University of Toronto,Toronto, ON, Canada6Toronto General Research Institute,University Health Network, Toronto,ON, Canada

The Rise of Microphysiological SystemsIn tissue engineering, assays reliant on 2D cellular monolayers are utilized to decipher the mech-anisms of action for drugs or other external stimuli such as atmospheric composition [1–3]; how-ever, these assays cannot be extrapolated to predict native behavior in response to the samestimuli [4,5] for example, screening the efficacy of a drug in a monolayer metabolic activityassay will not reflect the response of native 3D tissue to those same drug doses; monolayers sim-ply lack too many of the properties of the native niche. Not only do they lack biochemical and at-mospheric gradients [6], but stiff tissue culture plastic causes cells to lose their original phenotype[7]. The monolayer organization prevents cells from experiencing the appropriate cell–cell andcell–extracellular matrix (ECM) interactions [8]. However, despite these limitations, 2D culturesare used routinely as they require extremely low reagent volumes [9], and are compatible withexisting ‘omics’ and high-content analysis platforms [2,8,10].

To overcome these limitations of 2D cultures, a series of 3D model platforms have beendeveloped. These include, but are not limited to, cell sheets,microtissues (see Glossary), matrixencapsulation, and biofabricated constructs [11].

Cell sheets are confluent 2D monolayers recovered with the ECM intact as a single sheet fromtemperature-responsive [12] or fibrin-coated dishes [13], whereby thick constructs can beformed by stacking multiple sheets. However, the challenge of fabricating thick constructs of clin-ically relevant size often results in the development of necrotic regions due to a lack of nutrienttransport and oxygen diffusion [14]. One possible approach to avoid necrotic regions was tovascularize the cell-sheet constructs, as demonstrated for cardiac tissue [15]. By coculturing en-dothelial cells (ECs) and cardiac cells, vascular networks formed within each sheet. After recoveryand stacking, the multilayered constructs resolved nutrient and oxygen transport within theconstruct, as well as provided the support of an ex vivo vascular bed to adjacent tissue.

Trends in Biotechnology, Month 2019, Vol. xx, No. xx https://doi.org/10.1016/j.tibtech.2019.04.009 1© 2019 Elsevier Ltd. All rights reserved.

https://doi.org/10.1016/j.tibtech.2019.04.009




7The Heart and Stroke/Richard LewarCentre of Excellence, Toronto,ON, Canada8https://www.otago.ac.nz/christchurch/departments/orthomsm/people/khoon-lim.html9https://www.bzhanglab.com10https://www.labs.chem-eng.utoronto.ca/radisic/11https://www.otago.ac.nz/regenerative-medicine/index.html

*Correspondence:[email protected](T.B.F. Woodfield).@Twitter: @Khoonono (K. Lim),@bzhang7 (B. Zhang), @milicaruoft(M. Radisic), and @Tim_WoodfieldNZ(T.B.F. Woodfield).


The development of microtissue models relies on self-assembly of cells and their native ECM tocreate dense aggregates (also termed spheroids or organoids) smaller than 300 μm. Thesemicrotissues can be formed from one or more cell types (cocultures) in large quantities using ul-tralow attachment or hanging-drop microplates [16]. Compared with monolayers, microtissuesallow cell–cell and cell–ECM interaction in a 3D environment and permit self-organization in re-sponse to cues from biochemical and atmospheric gradients. This self-organization, however,leads to heterogeneity and the presence of the necrotic core, which may be detrimental toassay robustness [17]. Microtissues offer the ability to observe tissue-level behavior, importantlyin a format compatible with imaging-based high-content screening and analysis with fluoro-chromes or genetically modified cells [8].

An additional commonly adopted 3Dmodel platform employs an exogenous ECM to encapsulatesingle- or multiple-cell suspensions in a synthetic or naturally derivedmatrix (or hybrid thereof) typ-ical in hydrogel form [18]. Research into exogenous ECM, or hydrogel, development started in the1960s [19] and is still advancing rapidly [20]. Briefly, this ECM composition may be synthetic (e.g.,polyethylene glycol [21]) or naturally derived [22] from plant (e.g., alginate [23]), animal (e.g., silk[24,25], gelatin [26], collagen [27]), or recombinant (tobacco collagen [28], bacterial collagen[29]) sources as necessary to satisfy particular tissue requirements. Furthermore, the ability to tai-lor the electrical/mechanical properties [30,31] and functional properties [32,33] has permitted re-searchers to investigate a variety of applications, including musculoskeletal [34–36] tissueengineering and cancer research [37–39]. However, despite the broad selection and customiza-tion of hydrogels, they cannot be assumed to recreate all of the native ECM aspects, such as thepresence of appropriate adhesion ligands and proteins as well as the resulting structural organi-zation [5].

Biofabrication [40] of complex 3D tissue constructs in which combinations of cells [41], ECMcomponents (i.e., bioink [32,35,42,43]), and support structures (e.g., scaffolds for bone [44])are spatially organized (e.g., for neural [45], vascular [46], or tumor [47–49] targets) in apredetermined manner [50,51] has advanced rapidly and is now an established field [52–56].These multimaterial hybrid constructs [57] are part of a trend to control the spatial organizationover multiple cell types to achieve a more realistic recapitulation of the native tissue organization[58]. This degree of automation and control has enabled 3D cell-based assays to be executedwith the reliability and robustness required to advance the technology toward validated replace-ments for animal testing (https://ntp.niehs.nih.gov/pubhealth/evalatm/accept-methods/index.html) [48,59].

At peak complexity, are MPSs [60]. MPSs combine the biological complexity of spatially orga-nized tissue models, through biofabrication, with the technical complexity of a supporting envi-ronment tailored to replicate aspects of the tissue model’s native niche. MPS are uniquelypositioned to examine systemic responses by combining multiple tissue or organ compartmentsin bioreactors providing native-like stimuli (e.g., mechanical compression in an articular jointmodel, fluid shear stress in a vascular tissue model) under monitored conditions [60]. A numberof articles and reports from academia, the pharmaceutical industry, and government agenciessuggest that MPSs will provide the best opportunity to predict in vivo responses using anin vitro assay [1,60–63], but a multitude of obstacles are hindering development.

In the technical domain of the MPS (i.e., the bioreactor), the obstacles are associated with biore-actor material selection to avoid compound absorption, maintaining sterility, incorporating bubbletraps and in-line sensors, and providing each tissue module with the correct flow rate despitebeing connected in series [64]. Also, the tissue modules must permit the appropriate allometricconsiderations in terms of proportional tissue and medium volumes [65,66]. These technical

2 Trends in Biotechnology, Month 2019, Vol. xx, No. xx

https://ntp.niehs.nih.gov/pubhealth/evalatm/accept-methods/index.html

https://ntp.niehs.nih.gov/pubhealth/evalatm/accept-methods/index.html

[email protected]

GlossaryBioassembly: ‘The fabrication ofhierarchical constructs with a prescribed2D or 3D organization throughautomated assembly of pre-formedcell-containing fabrication unitsgenerated via cell-drivenself-organization or through preparationof hybrid cell-material building blocks,typically by applying enablingtechnologies, including microfabricatedmolds or microfluidics’.Biofabrication: ‘The automatedgeneration of biologically functionalproducts with structural organizationfrom living cells, bioactive molecules,biomaterials, cell aggregates such asmicro-tissues, or hybrid cell-materialconstructs, through Bioprinting orBioassembly and subsequent tissuematuration processes’.Biofabrication line: a collection oflaboratory equipment employed toconvert computer models, cellularmaterial, and acellular material into oneor all components of amicrophysiological system in amaximally automated manner.Bioink: ‘A formulation of cells suitablefor processing by an automatedbiofabrication technology that may alsocontain biologically active componentsand biomaterials’.Bioprinting: a subset of biofabricationtechnologies using computer-aidedtransfer processes for patterning andassembling living and nonliving materialswith a prescribed 2D or 3D organizationto produce bioengineered structuresserving in regenerative medicine,pharmacokinetic, and basic cell biologystudies.Body chip, body plate: a body chip isa bioreactor housing a collection oftissues (or tissue/organ constructs)connected by one or more fluid circuits.A body plate is a device with multipleonboard replicates of a body chip.Hybrid construct: a tissue constructcomprising multiple bioinks orbiomaterial inks fabricated using one ormore techniques.Microphysiological system (MPS):‘a microfluidic device capable ofemulating animal biology in vitro at thesmallest biologically acceptable scale,defined by purpose’.Microsphere: in the field ofbiofabrication, a microsphere is acollection of cells encapsulated inspheroid of exogenous extracellularmatrix (typically a hydrogel), either


challenges may be summarized as a need to increase system complexity to accommodate targettissues and monitor medium conditions for temporal assays, all while maintaining compatibilitywith external analysis platforms and imaging modalities.

In the biological domain (i.e., the tissue construct), advanced biofabrication techniques [58] maybe employed to incorporate vasculature [67], innervation [68], and other local (e.g., mural [69])and systemic cell types (e.g., immune [70]). The target niche must be fully characterized withthe biochemical, electrophysiological, and atmospheric gradients refined to match accordingly[71]. Until the biological and technical challenges are resolved, systems seeking to increase sam-ple numbers must sacrifice capabilities (e.g., perfusion, mechanical stimuli), resulting in an appar-ent complexity–throughput dichotomy when examining the MPS in the field. This reviewdiscusses these challenges to MPS development and proposes collaborations to expedite theresolution of these challenges in the context of a complete MPS experimental workflow: anMPS-terminated biofabrication line.

Balance of Biological Complexity and Throughput in MPS DevelopmentCurrent MPSs in Terms of Chips and Plates, Tissues and BodiesIn the context of MPSs, experimental throughput is a function of the number of independentmodel replicates residing on a single platform and the speed of tissue production and data acqui-sition. By contrast, model complexity is a function of the number of interconnected tissues in eachmodel, the inclusion of biomechanical stimuli, the presence of high-level tissue architecture, andthe integration of on-chip sensors. However, to better understand the relationship betweenthroughput and complexity, we quantified the two factors by limiting our consideration to thenumber of independent models on a single platform and the number of interconnected tissuesin each model, respectively (Figure 1). Based on these criteria, we have examined and summa-rized ten different representative state-of-the-art technologies in the field and review potentialtrade-offs between captured complexity and throughput. These technologies with supportedsample dimensions are provided in Table 1.

A step up in complexity from tissue chips are body chips, which are multiple tissue chips linkedin a continuous fluid circuit. The TissUse/ProBioGen body-on-a-chip systems (TissUse)employed on-chip micropumps to transfer media to four tissues either as a two-tissue circuit induplicate as demonstrated for discs of cast cell-laden hydrogel [72] or in series as a four-tissuecircuit populated with an intestinal tissue model, an infant skin biopsy, 20 liver microtissues,and proximal tubule cells seeded onto a membrane [73]. Whereas the TissUse system imple-mented on-chip micropumps, the two-tissue Homunculus implemented electrodes and real-time fluorescent sensors [74]. A larger variant targeted a six-tissue absorption, distribution, me-tabolism, and excretion (ADME) pathway.

The CNBio Single/Multi Organ-on-a-Chip™ plates (CNBio) operated according to a similar prin-ciple, adding docking-station capabilities to maintain a microplate footprint while supporting up to12 tissue scaffolds as single-tissue body replicates [75,76] or interconnected in multitissue cir-cuits as demonstrated with tissue scaffolds and Transwell® inserts [77]. The Draper HumanOrgan System™ (Draper HOS) permitted cultivation of up to 12 tissue modules. Under the USNational Institutes of Health (NIH) Tissue Chip for Drug Screening program, the Woodruff labora-tory utilized the system as a base for a female reproductive platform (EVATAR) supporting tissuesin Transwell® inserts [73].

Moving on to body plates, or body chips with on-plate replicates, the University of Pittsburg(UPitt) osteochondral platform, supported by the NIH Tissue Chip program, was designed to re-capitulate the bone–cartilage region of the articular joint and was based on a mechanically

Trends in Biotechnology, Month 2019, Vol. xx, No. xx 3

natural or synthetic, typically used asbuilding blocks in bioassembly. In thisreview, the same spheroid without cellsis a cell-free microsphere.Microtissue: a collection or aggregateof cells (typically spheroid shaped) with ahigh degree of cell–cell contact, boundby secreted autologous extracellularmatrix. Prior to unification of terminologyby the International Society forBiofabrication, microtissues were alsoknown as spheroids or cell aggregates.Tissue chip, tissue plate: a tissue chipis a bioreactor housing a single tissue/organ construct. A tissue plate is adevice with multiple onboard replicatesof a tissue chip.


stimulated dually perfused tissue (osteogenic medium and chondrogenic medium). The total plat-form capacity as specified in the introductory article was 96 tissue constructs divided into 12eight-tissue bodies [78,79]. Theoretically, these eight-tissue bodies could be placed in series toform a larger multitissue body as the system utilized external pump and tubing to connect thesample housing to reservoirs.

A recently described body plate bioreactor platform for culture of parenchymal and barrier tissueconstructs under active perfusion was designed to marry high complexity with high throughput(UOtago) [80]. The external pump and the ability to individually address two fluid circuits in eachof the 96 wells permitted arbitrary tissue combinations between 192 one-tissue bodies and a sin-gle 192-tissue body. In contrast to common MPSs utilizing external peristaltic pumps, systemicpriming volume was minimized by implementing 24-channel peristaltic pumpheads in a micro-plate footprint between the sample housing and the microplate media reservoirs. The platformwas demonstrated for use with hydrogel-based vascular constructs and bioassembled ovariancancer models.

The last classification, tissue plates, is arrays of tissue chips for on-plate replicates. The 4DesignBiosciences Vascularized Micro-Organ Platform™ (4Design) is an example of a microplate de-signed for arrays of independent tissues (one-tissue bodies), as described in the literaturesupporting 12 modules with three tissues in series as replicates [81], while the commercializedversion supported 16 modules. The MIMETAS OrganoPlate™ (MIMETAS) supported up to 96tissues [82]. Perfusion in the 4Design and MIMETAS MPS was achieved via gravity-assistedmedia transfer as in the EU/FP7 BOCMPS. The EU (FP7-ICT) Programme ‘Body-on-a-Chip’ col-laborative project (BOC; grant agreement ID: 296257) developed a platform to perform hepato-toxicity testing on liver microtissues. The resulting platform arranged 66 microtissues into 11six-tissue bodies [83]. Further, more complex, configurations were not possible as perfusionwas achieved via gravity-assisted media transfer between on-chip reservoirs on either side ofthe tissue series [84].

Trends in MPS DevelopmentCloser examination of the above MPS platforms revealed the technical trends driving the com-plexity–throughput dichotomy. The systems focused solely on sample numbers (e.g., EU/FP7BOC, 4Design, MIMETAS) utilized the ANSI/SLAS microplate standards (ANSI/SLAS 1-2004through ANSI/SLAS 4-2004) and mainly implemented gravity-assisted perfusion in place of anactive pump. Additionally, these MPSs relied on small, thin tissue volumes with thicknesses be-tween 0.1 mm and 0.12 mm, thus remaining within the focal depth of high-content imaging(HCI) modalities [94]. These ensured compatibility with laboratory robotic and imaging systemsfor rapid handling and analysis.

The complexity focused systems – that is, those that permitted interconnections between tissuemodules (e.g., TissUse, Homunculus, CNBio, Draper, UPitt) – used active pumping. All systems(with the exception of UPitt and UOtago) implemented on-chip pneumatically driven pumps tominimize the priming volume of the medium circuit. The introductory articles for these MPSs per-formed detailed allometric or functional scaling to compare the achieved medium-to-tissue vol-ume ratios and perfusion rates with respect to their in vivo application targets (e.g., the Draper-based EVATAR system) [95]. MPSs not capable of achieving in vivomedium-to-tissue volume ra-tios would have a potential drawback given the dilution of soluble factors for cell signaling to theexterior of the same cell (autocrine), between cells locally (paracrine), and between tissues (endo-crine) [96]. With respect to the supported tissue models, the complexity-focused MPSs were de-signed to accommodate tissues in well inserts, either as part of the tissue construct (e.g., withhydrogel in UPitt [79], with cells in CNBio [77]), as a carrier (e.g., for microtissues in TissUse


Body

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s per

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9

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16, 4Design

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UPi OC

200

190

180

96

UOtago

192

Tissue plates Gravity-driven

11, EU BoC

90 10 9 1 2 3 4 5 6 7 8 20 30 40 50 60 70 80 100 190 180 200

Bioreactor throughput (# Replicates)

Future developments

10, TARA IV

Tissue chips

12

Body chips Internal pump

TissUse

Draper HOS 96, MIMETAS

TrendsTrends inin BiotechnologyBiotechnology

Figure 1. Interplay between Complexity and Throughput in Representative Microphysiological System (MPS) Bioreactors. A body is a fluid circuitcontaining one or more tissue models. Tissue chips with one tissue or tissue interface are represented by the black diamond on the plot with a representative chip [85].Each point represents a permissible configuration that has been assigned for the purposes of this review. For those systems with interconnected tissue/organmodules, lines connect these points to represent the entire complexity–throughput spectrum of that MPS. Systems with single points, such as those from InSphero,4Design, and MIMETAS, implemented on-plate replicates (tissue plates), which typically cannot be connected in series to form more complex systems, and thus havebeen assigned a point representing the right-most end of a horizontal line starting at 1. Tissue/organ-on-a-chip modules representing a single body comprising a singletissue are at the lower left of the plot (black diamond). At the bottom left are commercial multitissue body platforms. At the bottom right of the plot are microplatesdesigned for arrays of independent tissues (one-tissue bodies). The plot reveals a trend in the perfusion mechanisms where high-throughput MPSs use gravity-drivenperfusion and high-complexity MPSs use pump-driven perfusion. Platform data from: 4Design [81], CNBio [75,77], Draper HOS [87,88], EU BOC [86], MIMETAS [89],TARA IV (InVade) [90], TissUse [73,91], UOtago [80], and UPitt OC [78,92]. Images adapted, with permission, from the respective articles. Image for EU BOC obtainedfrom InSphero website (https://web.archive.org/web/20160401003403/https://www.insphero.com/company/eu-project-the-body-on-a-chip).



Image of Figure 1

https://www.insphero.com/company/eu-project-the-body-on-a-chip

Table 1. Sample Geometries Utilized in Current MPS Bioreactors

MPSbioreactor

Classification Body complexity(max. # tissues/circuit)

Bioreactor throughput(max. # replicates)

Perfusionmethod

Largest sample size Refs

4Design Tissue plate 1 16 Gravity driven 1 mm × 2 mm × 0.12 mm [93]

EU BoC Tissue plate 6 11 Gravity driven 200 μm to 300 μm [83]

MIMETAS Tissue plate 1 96 Gravity driven 4.5 mm × 0.2 mm × 0.12 mm [82]

TARA InVade Tissue plate 2 10 Gravity driven 4 × 3 × 2 mm [90]

CNBio Body chip 12 10 Internal pump Ø15-mm tissue scaffolds, Ø12-mmTranswell® inserts

[77]

Draper HOS, EVATAR Body chip 12 10 Internal pump Ø9-mm Transwell® insert [73]

TissUse Body chip 4 2 Internal pump Ø6.5 mm × 5.6-mm mass, Ø9-mmTranswell® insert

[72]

UOtago Body plate 192 192 External pump Ø5.5 mm × 10 mm [80]

UPitt Body plate 96 10 External pump Ø3 mm × 6 mm [78]


[73], for primary tissue in EVATAR [88]), or as a supplied model (e.g., MatTek EpiIntestinal™ inTissUse [73]).

In general, the advantages of gravity-driven flow include simplicity of operation and ease of re-moving bubbles in open-well-plate configurations, targeting users such as scientists in pharma-ceutical companies who could use the platform for drug discovery and disease modeling. Aregular work flow in pharma includes screening in well plates andHCI in machines that accommo-date well plates as well as fluid handling by pipetting robots and ultrasonic fluid dispensers. It isimportant to consider that pumps have not been included in a regular pharma workflow. Thedrawbacks of gravity-driven flow include a limited range of flow rates available in any single de-vice, inability to achieve pulsatile flow, and transient application of perfusion. Flow driven by sy-ringe or peristaltic pumps provides a solution for these limitations, albeit at the expense ofrequiring specialized equipment and the incorporation of bubble traps.

Last, the MPSs considered in Figure 1 do not include on-chip sensors, with the exception ofthe Homunculus [97]. However, given developments with single-tissue chips, future devicesmay monitor cellular activity across a spectrum of technologies [98]. A recent full-featuredcollection of modules implemented a suite of electrochemical, physiological, and biochemicalsensors to monitor and assay conditions [85]. Other systems have implemented functionalizedelectrodes to quantify glucose and lactate concentrations in microtissue cultures [99] andan in-line ELISA to quantify hepatocyte albumin secretion [100], and mass-spectrometrycompatibility was implemented to measure soluble factors secreted by adipocytes [101] andJurkat T cells [102].

The UOtago platform focused on balancing complexity and throughput by adopting the ANSI/SLAS microplate format for both the sample housing and the medium reservoir to maintain com-patibility with standard microscopy and liquid handling platforms while retaining region-specificindependence of media in tissue models carried by well inserts (similar to those utilized byUPitt) [80]. In addition, the platform expanded noninvasive sample assessment to high-resolution computed tomography (e.g., μCT) via modular microplates and leveraged a dockingstation to permit in situ imaging during perfusion. For perfusion, reliance on an external peristalticpump caused the priming volume of each circuit to remain 10× higher than the near-physiologicalvalues achieved by the EVATAR system [95]; however, the docking station also permitted tissuemodule interconnections similar to the UPitt system in external tubing, but with an expanded



degree of flexibility by permitting an arbitrary number of tissues in series (1 to 192) with varyingdegrees of modulated crosstalk.

Over the past decade, government initiatives have supported the development of MPS technol-ogy, largely through the US NIH Tissue Chip [71] and EU/FP7 BOC programs (with AstraZeneca)[83]. The systems have reached a state of maturity to permit comprehensive validation initiatives.Future developments, such as the implementation of onboard sensor technologies, may facilitatethe translation of these body chips into body plates for high-throughput studies.

Opportunities for MPS-Terminated Biofabrication Lines at the Convergence ofBiofabrication Techniques and Bioreactor TechnologiesBiomechatronic design methodology correlates existing technologies and techniques to fulfill theend biological and technical requirements [103]; when applied specifically to MPS development,one may identify the rate-limiting steps of the experimental workflow and pinpoint the causes tothe complexity–throughput dichotomy discussed in the previous section. In Figure 2, theMPS ex-perimental workflow is portrayed as a biofabrication line to clarify the movement of cells and con-structs (blue arrows) through laboratory equipment (rectangles). Through this representation, thedegree of integration in tissue construct and bioreactor production (in the box labeled bioprinter)may be weighed against the overall workflow. For example, in the fully integrated situation wherethe bioprinter concurrently fabricates the bioreactor components and tissue construct to elimi-nate intermediate loading steps, as in a recently described liver chip [104], the labor savings inthe overall workflow alone may not warrant the imposed constraints on bioreactor materialsand fabrication method. This is be discussed in more detail at the build stage.

Stages 1 and 2: Design and PrepareKey steps in the design stage could involve the conversion of data from in silico studies investigat-ing basic biology and elucidating systemic interactions (Figure 2, in silico), prior data from in vitro/vivo studies, and known aspects of human and animal physiology (combined in Figure 2 as priordata) into a desired spatial organization of cell-laden (e.g., microtissues) and acellular (e.g., poly-mer) constituents of a tissue-engineered construct. At this point, the manufacturing methodswould be decided among one or more common techniques described in Figure 3 to achievethe desired approximations of the composition, organization, and niche of the target tissue ororgan achievable with the available fabrication modalities, materials, and cell sources. The con-struct setpoint would then be converted into a set of instructions for the bioprinter usingcomputer-aided drafting/manufacturing (CAD/CAM) software. Here execution speed would bedependent on algorithmic efficiency and IT infrastructure.

Key steps for the subsequent prepare stage include purification of cell populations, expansion ofthe purified populations, differentiation to the desired lineages, and, if necessary, the combinationof cells with additional, sterile, material such as ECM components and/or hydrogel. The resultingfeedstock for the next stage may include cell-laden bioink [57] or 3D cellular modules such asmicrotissues (dense collections of cells held together with autologous ECM), microspheres(cells encapsulated in exogenous ECM), or even microspheres encapsulating microtissues[109]. For tissues utilizing primary cells rather than cell lines, a population of donor cells, either pri-mary or the differentiated progeny of induced pluripotent stem cells (iPSCs), could be purified toisolate the populations with the phenotypes for the target tissuemodel (cell selector). For both pri-mary and immortalized sources, the cells may be expanded on microcarriers, tissue culture plas-tic, or hollow-fiber reactors to achieve the yield necessary to create a tissue with the desiredvolume and cell density (expansion system). Depending on the construct geometry, the cell pop-ulations would then bemixed with a macromer suspension in any cell culture vessel to form bioink


Construct design

In silico studies Prior data

Bioprinter

Cell selector

Design

Expansion system

Bioinkmixer

Livingmaterial

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Donor �ssue

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generator

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Data

MonitoringControlunit

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Finishing


Figure 2. Stages of a Biofabrication Line. Arrow key: black, workflow direction; gray, movement of bioreactor; blue, movement of all other physical entities such ascells or constructs; orange, flow of information; yellow, all forms of biochemical and energetic stimulation. The bioprinter details three possible degrees of integration ofbioreactor and tissue construct (bio)fabrication: decoupled, partially integrated, and integrated.



Image of Figure 2


(bioink mixer), seeded into microtissue-forming microplates andmatured (microtissue generator),and/or encapsulated in hydrogel and passed through a spray nozzle or multiphase microfluidicchamber and crosslinked to form microspheres (microsphere generator).

Previously described devices and technologies exist for each of the proposed stages above[110]; for example, the StemCell RoboSep™ for cell separation [111] and the SartoriusCompacT SelecT™ [112] or bespoke systems [113] to expand cell lines in a variety of culturevessels. Fluid handling stations paired with hanging-drop microplates can generate microtissues(e.g., PerkinElmer Zephyr®G3with InSphero GravityPLUS™ [114]), microfluidic systems can gen-erate microspheres (e.g., Dolomite μEncapsulator 1™ [115]), and generic fluid handling stationscan mix bioinks. When placed into the context of the convergence of the biofabrication and MPSfields, a high-throughput MPS supporting biologically complex tissue models will require anextensive prepare stage. The cell quantities to achieve the throughput will lengthen the expansionstep, while the increasing number of different cell types or 3D cellular modules will requireresearchers to spend longer periods of time interacting with and transferring material betweenmore pieces of equipment. Fortunately, the burden on the researcher may be alleviated by auto-mating and integrating the separate preparation steps in a single robotic laboratory cabinet suchas the BioNex Hive™ [116].

Stages 3 and 4: Build and CultureThe build stage can utilize additive manufacturing technologies to transform construct blueprintsinto a construct using a series of dots and fibers of possibly cell-laden bioinks, adopting the (bio)fabrication techniques as specified in the blueprint (Figure 3). The execution speed of the buildstage would be a function of the production speed and entity transference into and out of thebioprinter. These key factors would be influenced by the degree to which tissue constructbiofabrication and bioreactor fabrication are integrated. This degree of integration would strikea balance between expediency and the limitations of the available production techniques and res-olution, as well as bioink and (bio)material properties (Figure 4).

In a decoupled approach, the tissue constructs and bioreactor are manufactured separately. Thisapproach offers the greatest flexibility with regard to manufacturing techniques, keeping bothopen to future advances: the bioreactor can leverage proven methods for mass production(e.g., injection molding, computer-controlled machining) regardless of the incompatibility of thatmethod with biological entities and without constraining the choice of biofabrication technique;that is, as long as the final construct is structurally sound and compatible with pick-and-place(PnP) robotics. Likewise, construct fabrication itself may be decoupled, as in bioassembly[40], to combine techniques reliant on tightly controlled build conditions (e.g., melt electrospinningto create stiff scaffolds with high spatial resolution [117]). Additionally, considerable attentionmust be paid to the design of the bioreactor and construct temporal geometries to guaranteerobust anastomosis.

In terms of biofabrication speed, material loading and construct transfer to the bioreactor may beperformed manually by a technician. Few bioprinters (e.g., Brown University Bio-P3 [118], CyfuseRegenova, Advanced Solutions BioAssemblyBot, nScrypt 3Dn) specifically include PnP endeffectors to automate the latter, while at present only the BioAssemblyBot has the movementenvelope to handle both. In addition to the lack of automation, the biofabrication process itselfis extremely slow. The fastest known techniques (extrusion of bioink and stereolithographicprinting of bioresin [32]) have achieved volumetric deposition rates of only 0.5 cm3 min−1 [119].The complexity-focusedMPSs reviewed in the previous section utilized this decoupled approach.For example, the TissUse system [73] relied on prefabricated MatTek tissue models and ex vivotissue located in transwells.


Pulsed laser

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(F) Laser-assisted bioprin�ng

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(K) Granule-based medium-assisted bioprin�ng

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The flexibility of the decoupled approach is represented visually in Figure 5. This broad range ofcurrently permissible combinations of bioreactor fabrication and construct biofabricationmethods (black border cells) offers a promising route to achieve high-throughput MPS withhighly complex tissue constructs. In the future, the decoupled approach may become evenmore flexible through the adaptation of powder-bed technologies (3DP and SLS) tobiofabrication (Figure 5, red border cells), through the use of biopowder. Given that bioresinis a cell-laden SLA resin and bioink is a cell-laden ink for 3D plotting, biopowder is cell-laden3DP or SLS feedstock. Biopowder could be microspheres encapsulating cells in hydrogel,as currently produced by the Dolomite μEncapsulator 1™ [115]. In 3DP, biopowder wouldbe suspended in density-matched carrier fluid at a high powder:carrier ratio (a highly viscousemulsion) to ensure that the powder particles remained in contact yet were restricted fromfusing spontaneously. The printer head would deposit nanoscale volumes of high-viscosityexogenous ECM at particle interfaces, simultaneously forcing the carrier fluid out of theinterface and bonding the particles. Similarly, in SLS, the biopowder would be suspended inacellular photocrosslinkable resin. The laser would fuse neighboring particles by selectivelycrosslinking the resin between said particles. While this is not strictly sintering due to theadditional material, the neighboring particles of a powder bed are still fused through thecontrolled application of light. In short, future 3DP or SLS bioprinters may be applied to createthe cellular components of tissue constructs.

On the opposite end of the spectrum is the integrated approach, a specialized example of leanmanufacturing in which the construct and bioreactor are produced simultaneously. While thisapproach is logistically riskier due to the reduction of permissible pause points (e.g., the inabilityto manufacture bioreactors en masse via injection molding and store for later use), the constructand bioreactor geometries may be highly entangled. This high degree of entanglement, orintegration, reveals two important advantages: the complexity of the construct–bioreactorinterface may be increased and the material required for the structural integrity of softconstructs may be reduced, possibly increasing the sample density, hence throughput, inthe same bioreactor footprint.

Figure 3. Classes of Additive Manufacturing Technologies Commonly Adopted for Microphysiological System (MPS) Production Utilizing Acellular(Blue) (Bio)Material Feedstock (A, B, D, G, H, I, L [105], and C [106]), which Include Subsets of Biofabrication Technologies for Cell-Laden (Orange)Bioinks (E, F, J, K, M, N [107]) or Hybrid Combinations Thereof (O [108], P [109]). Images adapted, with permission, from the respective articles. To give aninsight into the various layer-by-layer fabrication approaches, each additive manufacturing (blue) and biofabrication (orange) technology can generally be assigned tofour technology classes:(i) Laser- or light-based technologies: These additive manufacturing technologies comprise: (A) Selective laser sintering (SLS) of powders; (B) laser stereolithography (SLA) onto

liquid resins; (C) digital light processing (DLP) of a projected mask of light onto liquid resins; and (D) high-resolution two-photon polymerization (2PP) of laser pulses into pho-tosensitive resins. The subset of biofabrication technologies includes: (E) lithography-based bioprinting that adopts SLA/DLP approaches combined with cell-laden photo-sensitive resins; and (F) laser-assisted bioprinting or laser-induced forward transfer (LIFT) that uses pulsed laser to selectively project cell-laden droplets onto a substrate.

(ii) Extrusion-based technologies: These additive manufacturing technologies comprise: (G) fused deposition modeling (FDM) of a (bio)material filament spool extruded from aheated nozzle; (H) extrusion 3D plotting (3DF) of a molten (bio)material under pressure or displacement from a heated nozzle; and (I) solution electrospinning of randomly ar-ranged (bio)material/solvent nanofilaments from high-voltage spinneret onto a collector or melt electrowriting (MEW) of ordered molten (bio)material microfilaments from high-voltage heated spinneret onto a computer-controlled x-y-z collector. The subset of biofabrication technologies includes: (J) extrusion 3Dbioprinting of cell-laden (shear thinning)bioink under pressure or displacement from a temperature-controlled nozzle often followed by subsequent photocrosslinking; and (K) granule-based medium-assistedbioprinting of cell-laden bioink into a granule-based or buoyant bath or support medium.

(iii) Jetting- or powder-based technologies: These additive manufacturing technologies comprise (L) 3D printing (3DP), combining inkjetting of droplets of a binder material onto a(bio)material powder bed. The subset of biofabrication technologies includes: (M) inkjet bioprinting using thermal or piezoelectric jetting head to jet cell-laden bioink droplets ontoa surface; and (N) multijet bioprinting, similar to inkjet bioprinting with multiple jetting heads and multiple cell-laden bioink droplets.

(iv) Hybrid biofabrication technologies: These rapidly emerging technologies utilize combinations of the above additive manufacturing and/or biofabrication technologies to createmore complex hybrid constructs or 3D tissue models. For example: (O) hybrid 3D bioprinting of molten (bio)material filament (or other light- or powder-based technologiesabove) withmultiple cell-laden bioink filaments via extrusion, typically usingmultiple combinations of temperature-controlled extrusion nozzles as well as (shear thinning) bioinkswith different cells; and (P) 3D bioassembly of tissue spheroids that can involve combinations of extrusion 3D bioprinting of molten (bio)material filament (or other light- or pow-der-based technologies above) with automated (bio)assembly of tissue spheroids/organoids or cell-laden bioink spheroids typically fabricated using high-throughputmethods,whereby each spheroid can contain different cells and/or bioink formulations.


Bioprinter

Living material

Construct blueprints

Inanimate material Bioreactor

Decoupled Par ally integrated

Integrated


Figure 4. Degrees of Integration for Tissue Construct and Bioreactor Production with Examples of Decoupled [92], Partially Integrated [55], andIntegrated [104] Methods. Graphics adapted, with permission, from the respective articles.


Emerging examples of this approach (Figure 5, gray cells) rely on nozzle-dispensing print technologies(e.g., 3D Plot, ES, FDM) and/or component manipulation via PnP (MA). Specifically, recentstudies created individual perfused tissue models targeting liver [104,120], cancer [121], andvascularized kidney [122]. It has also been applied to skin [123] and neural [124] constructsfor static (nonperfused) well-plate cultures. Current research into novel biomaterials suitablefor biofabrication feedstock [57] ensures that researchers have an expanding pallet withwhich to satisfy MPS structural design requirements while mitigating exposures to cytotoxicvolatiles Conversely, utilization of volumetric technologies to increase print speed (e.g., DLP)or metals to increase mechanical strength (e.g., sintered titanium) is unlikely given the relativeincompatibility of fabrication conditions. The future for this approach (Figure 5, red cells) maybe to leverage the increased printing resolution of existing planar-dispensing biofabricationtechnologies (e.g., Jet, LAB) to further increase the complexity of the construct–bioreactorinterface or to create smaller features in either component.

Between decoupled and integrated, a partially integrated approach allows the fabrication of theconstruct directly inside the bioreactor. It permits soft or amorphous constructs and facilitatesmechanical coupling between the construct and mechanical stimuli (e.g., seeding cells onto amembrane to undergo a tension regime to mimic a lung [125]). From an execution speed per-spective, this technique has the advantage of permitting mass production methods for bioreactorfabrication and also removes the construct transfer step; however, it still imposes constraints onthe bioprinter printheads (e.g., an extrusion nozzle or jetting head must reach the bottom of thesample chamber in the bioreactor). Thus, while the bioreactor may be fabricated with any tech-nique, construct biofabrication is limited to nozzle-dispensing and PnP-based techniques


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Future capability


Figure 5. Degrees of Integration as Related to (Bio)Fabrication Technologies. Terms in alphabetical order as defined in [119] where appropriate: 2PP, two-photonpolymerization; 3DP, 3D printing; 3D Plot, 3D plotting of extruded material – includes PAM; Cast, any technique reliant on intermediate components such as a disposablemold – includes injection molding; DLP, digital light processing; ES, electrospinning; FDM, fused deposition modeling – also known as 3DF; Jet, all jet printing includinginkjet and valve-based; Jig, a build platform contacting the component on more than one surface, which includes a machine chuck or removable mold; LAB, laser-assisted bioassembly – also known as LIFT and MAPLE; MA, general modular assembly – includes bioassembly with spheroid-based approaches such as Kenzan;PAM, pressure-assisted microsyringe dispensing and wet-spun technologies; PnP, pick-and-place robotics; SLA, stereolithography; SLS, selective laser sintering;Sub, the broad field of subtractive machining technologies including laser ablation.


(Figure 5, black-patterned cells). With the advent of bioresins [32,126], modified SLA, DLP, orprinters may facilitate the inclusion of these techniques with open-top bioreactor geometriesclear of internal opaque obstructions (Figure 5, red-patterned cells). Likewise, the introductionof new tomographic DLP printing technology [127] may accelerate development of a light-based partially integrated approach, provided the bioreactor materials are transparent to the rel-evant wavelengths [128,129]. Therefore, in the future, mass-produced bioreactors may be com-bined with the most rapid high-resolution biofabrication methods resulting in a streamlined MPSbiofabrication line centered around constructs with increased physiological relevance.

Last, an MPS is formed once a construct is located inside the bioreactor and ancillary control andmonitoring components are connected. Final MPS complexity is a function of complexities in thetechnical and biological domains. An increase in biological complexity (i.e., vertical movement


Image of Figure 5

Outstanding QuestionsCan we overcome the trade-offs be-tween the complexity of 3D biologicalmodels and the experimental through-puts and solve the complexity–through-put dichotomy?

Can we achieve greater flexibility in thebiological model in accordance with ex-perimental needs by integrating or par-tially integrating the fabrication of tissueconstructs with the design of bioreactorsand MPSs?

Canwe further improve the biological rel-evance of our engineered tissue modelswith the routine use of primary patient-specific cells or induced pluripotentstem cells?


toward future directions in Figure 1) may be considered as the drive to create physiologically rel-evant tissue constructs. Similarly, the technical domain (i.e., lateral movement toward future di-rections in Figure 1) encompasses efforts to perform the following for an ever-increasingnumber of samples: generate and analyze data to maintain construct health (e.g. pH, dissolvedoxygen and metabolite levels), observing the impact of induced stimuli of interest (e.g., paracrinelevels, localization of damage-sensing fluorescent markers) and the degree to which automationis used to perform routine tasks (e.g. to apply stimuli and refresh culture media).

Concluding Remarks and Future DirectionsMicrophysiological systems are emerging platforms to predict in vivo behaviors bymeans of in vitroexperimentation. However, further developments are necessary before the systems can achieveboth high model complexity representative of human physiology and high experimental through-puts, resulting in the current complexity–throughput dichotomy. In the short term, the context ofthe biofabrication line may be used to guide the design of tissue models and bioreactors tomatch the tissue requirements (e.g., mechanical coupling, intact removal for histological assess-ment) to the implemented construct–bioreactor production integration approach. A decoupled ap-proach offers an opportunity to utilize the widest range of biofabrication methods to maximize bothproduction speed and construct complexity, with the notable requirement that the resulting con-struct must survive transfer into a receptive MPS. The integrated approach overcomes this needfor structural integrity thereby permitting constructs closer to their target in vivo tissue. However,in this approach fewer techniques for construct and bioreactor production are interoperable.

One potential future direction in biological modeling is the creation of vascularized functionalorganoids where dynamic perfusion can be applied through an internal vasculature integratedwithin a sophisticated stem cell-derived organoid. Current solutions so far all adopt a decoupledapproach. Because of the specific requirement of the biomatrices and the complex differentiationprotocol, organoids are grown separately and then transferred to an organ-on-a-chip device withan open-well design [130,131]. Although we have seen impressive success in this area, to estab-lish perfusable vasculature that will fully integrate with organoids, a partially integrated approachmight be necessary to seamlessly integrate organ-on-chip bioreactors with the organoid differen-tiation and maturation process. This strategy could avoid the need to transfer fragile organoid tis-sues, which introduce arbitrary interruption in the tissue development process. Future advancesin bioresin formulations that are more suitable for stem cell culture and bioprinter hardware mayposition the partially integrated approach as an attractive compromise between decoupled andintegrated (see Outstanding Questions). The corresponding MPS-terminated biofabrication linecould be automated, employ rapid high-resolution biofabrication technologies, and utilizemass-produced bioreactors to stimulate and monitor an array of near-physiological tissue con-structs, thus resolving the complexity–throughput dichotomy. It is also important to note thatthe application of producing large arrays of complex functional tissues and maturing them on-chip is not restricted to drug testing or fundamental biological studies. If a platform allows tissueextraction for downstream analysis via a partially integrated approach, the same capability willalso enable tissue extraction for implantation with a modular tissue engineering approach. Tissueengineering in drug discovery and regenerative medicine are just two sides of the same coin.

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