Design and Fabrication of the Vertical-Flow Bioreactor for Compaction Hepatocyte Culture in Drug Testing Application

biosensors

Article Design and Fabrication of the Vertical-Flow Bioreactor for Compaction Hepatocyte Culture in Drug Testing Application

Liang Zhu 1,2,3, Zhenfeng Wang 2, Huanming Xia 1,4 and Hanry Yu 2,3,5,6,*

1 Singapore Institute of Manufacturing Technology, Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, #08-04 Innovis, Singapore 138634, Singapore; [email protected] (L.Z.); [email protected] (H.X.) 2 Mechanobiology Institute, National University of Singapore, T-Lab, #05-01, 5A Engineering Drive 1, Singapore 117411, Singapore; [email protected] 3 Institute of Biotechnology and Nanotechnology, Agency for Science, Technology and Research (A*STAR), The Nanos, #04-01, 31 Biopolis Way, Singapore 138669, Singapore 4 School of Mechanical Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei St., Nanjing 210094, China 5 Department of Physiology, Yong Loo Lin School of Medicine, MD9-04-11, 2 Medical Drive, Singapore 117597, Singapore 6 Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #10-01 CREATE Tower, Singapore 138602, Singapore * Correspondence: [email protected]

Abstract: The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-ﬂow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance Citation: Zhu, L.; Wang, Z.; Xia, H.; of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a Yu, H. Design and Fabrication of the Vertical-Flow Bioreactor for functional drug testing platform through the sustained maintenance of model cells. Compaction Hepatocyte Culture in Drug Testing Application. Biosensors Keywords: microﬂuidics; bioreactor; perfusion culture; hepatocytes; drug testing; design; fabrication 2021, 11, 160. https://doi.org/ 10.3390/bios11050160

Received: 5 April 2021 1. Introduction Accepted: 6 May 2021 With the development of in vitro culture of hepatocytes since the 1980s, there have Published: 19 May 2021 been numerous models and reactors created, catering to various applications such as the bio-artificial liver (BAL), drug toxicology testing, therapeutic development, and tissue Publisher’s Note: MDPI stays neutral engineering [1–3]. Due to high metabolism and detoxification properties of hepatocytes, it with regard to jurisdictional claims in is preferable to use a perfusion culture system for adequate nutrition and oxygen supply published maps and institutional affil- as well as fast waste removal [4,5]. However, the shear stress generated by perfusion may iations. damage the functionality or even the viability of hepatocytes [6,7]. An essential component of a perfusion bioreactor is therefore the shear stress filter, which minimizes the shear stress when the perfusate passes by the cells while still maintaining the level of mass exchange. The 3D-µFCCS our laboratory has developed made the first step in achieving this Copyright: © 2021 by the authors. balance by integrating pillar filters around the cell seeding channel so that the loading Licensee MDPI, Basel, Switzerland. cells may be well trapped and so that the shear stress may be “filtered” out during the This article is an open access article perfusion later on [6,8]. On top of that, there is an improved protocol by incorporating distributed under the terms and compaction force on top of perfusion culture to achieve better hepatocyte performances [9]. conditions of the Creative Commons However, the problems associated with the pillar filter in a cross-flow perfusion—the mass Attribution (CC BY) license (https:// transfer inefficiency [10], low throughput [8], uncontrollable manual compaction [9], limited creativecommons.org/licenses/by/ accommodation of cells [9], etc., still remain unsolved. We have thus reported previously 4.0/).

Biosensors 2021, 11, 160. https://doi.org/10.3390/bios11050160 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, 160 2 of 13

that the adoption of a nano-pore size membrane as the shear stress filter in a bioreactor allows for a buildup of pressure in the culture chamber, and leads to a “compaction” culture of the hepatocytes, which maintains a 3D morphology in a monolayer culture setup, enhancing the cell polarity and other hepatic functions [11]. This compaction culture is realized in a vertical-flow bioreactor (VfB) we made. In order to showcase the methodology used to design, fabricate, and test this unique VfB as a drug-testing platform prototype, this paper elaborates on the principles of the design, the methods of the experimental setup and optimization, as well as the hepatic function test, while using the double-well VfB as a case study. Notably, the VfB utilizes modular culture chambers which are scalable, ranging from a single well reactor to a 24-well array, increasing its flexibility in adapting the throughput, and reducing the cost of the VfB-based drug testing product to fulfill different application requirements.

2. Materials and Methods 2.1. Bioreactor Design and Fabrication The VfB was designed using Solidworks 2012 (Dassault Systèmes SolidWorks Cor- poration, Waltham, MA, USA) and AutoCAD (Autodesk, San Rafael, CA, USA). The computational fluidic dynamic (CFD) simulation was completed by ANSYS-CFX (ANSYS, Inc., Canonsburg, PA, USA). The main body of the VfB is fabricated by the thermoplastic polymethyl methacrylate (PMMA), which is biocompatible, transparent, and suitable for scaled-up production when the VfB transits to the industrialization stage [12,13]. Thermo- bonding was used for the assembly of the VfB, either by hydraulic pressure thermo-bonder (Atlas, Specac, Orpington, UK), or in a high-temperature oven (Modell 100–800, Memmert, Schwabach, Germany). Prior to bonding, the PMMA VfB layers were vacuum dried in a vacuum oven (VOS-301SD, Eyela, Singapore). All bonding work was done in a Class 1000 clean room in order to control the clean environment to ensure bonding strength. The double well VfB has two independent chambers linked with perfusion tubing and valve fixed onto a PMMA jig. Each chamber of the VfB utilizes the same size of that in a commercially available 24-well culture plate (Nunc®, Thermo, Waltham, MA, USA). The vertical-flow perfusion design uses the nano-pore-size membrane in Polyester (PET, Transwell®, Corning, Corning, CA, USA) as both the shear stress filter and the cell seeding membrane.

2.2. Cell Seeding and Culture Fresh primary hepatocytes were isolated from the male Wistar rats (250–300 g). William’s E medium was used as culture media, (Sigma, St. Louis, MO, USA) supple- mented with 1 mg/mL bovine serum albumin (BSA) (PAA Laboratories, Pasching, Austria), 10 ng/mL epidermal growth factor, 0.5 mg/mL insulin, 5 nM dexamethasone, 50 ng/mL linoleic acid, 10 units/mL penicillin, and 10 mg/mL streptomycin. The hepatocyte suspension was gently mixed and pipetted onto the collagen-coated cell seeding membrane in a spot-by-spot way so as to make the cell suspension reside on the membrane evenly. The hepatocytes were then maintained either in perfusion, static, or collagen sandwich ◦ culture (as control) in the CO2 incubator with 5% CO2 and 95% humidity at 37 C. For the sandwich culture, an overlay of 0.3 mg/mL collagen was added onto the cells the following day. The animals used in the experiments were handled under the approval of the Institu- tional Animal Care and Use Committee (IACUC) of the National University of Singapore (NUS). No human samples or participants were used, and no other ethics approval is required.

2.3. Hepatic Function Test Perfused culture media were collected daily for each chamber of the bioreactor and kept at −20 ◦C until the assay was carried out. The control was the supernatant of the static cultured hepatocytes on a 24-well culture plate. Urea production was quantiﬁed by BUN Biosensors 2021, 11, 160 3 of 13

urea nitrogen test (Stanbio Laboratory, Boerne, TX, USA). The CYP level was quantiﬁed on the CYP speciﬁc metabolites, with the internal standards (Cyp1a2, Cyp3a2: 100 ng/mL Acetaminophen-D4; Cyp2b2: 50 ng/mL OHbupropion-D6), by LC/MS measurement (LC: 1100 series, Agilent, Singapore; MS: LCQ Deca XP Max, Finnigan, Singapore). All functional data were normalized to the seeded cell numbers, which were determined by the PicoGreen assay (Molecular Probes, Eugene, OR, USA).

3. Results and Discussion 3.1. Design and Principles An ideal bioreactor for in vitro cell culture device, regardless of static or perfusion culture, should be constructed with nontoxic and biocompatible materials [14], preferably with optical transparency for cell status monitoring [15]. Each culture well or chamber needs to provide a large surface area for a sufficient number of cells to attach [14], high mass transfer efficiency [14], and preferably with flexible configuration to allow for co- culture, cell–cell signaling assays, etc. [15]. The culture device needs to be easy to assemble, allow for the easy insertion and retrieval of scaffolds or substrates, and with scalability for high throughput culture [15]. When it comes to a perfusion bioreactor, additional requirements arise such as providing a leak-proof culture chamber or well to allow for a medium flow perfusion with controllable flow types and rates to avoid the negative impact caused by the formation of air bubbles [15,16]. To maintain a good microenvironment of the cells under the perfusion culture, the perfusate needs to be evenly distributed across the large cell attachment surface, with sufficient culture medium replenished in-time and shear stress minimized [11]. Additionally, if the bioreactor is able to mimic the in vivo microenvironment during cell culture through maintaining the cells in 3D morphology or keeping the cells under a similar pressure as found in vivo, both the cell functions and the viability are expected to be enhanced and maintained for a sustained period of culture [11]. The VfB was designed to meet with the aforementioned features of an ideal perfusion bioreactor, with the unique “vertical-flow” perfusion to realize the compaction culture, and thus mimicking the in vivo microenvironment for cells [11] (Figure1), while avoiding the concentration gradients of culture media caused by the cell adsorption in the front of the perfusion path in many commonly seen “horizontal-flow” microfluidic bioreactors [6,8,17] (Figure1). If the perfusion path is horizontal across the culture chamber of 15 cm diameter, the media solvents will be absorbed by the cells along the path, resulting in a reduction of the media concentration while the flow length (and hence the cell-media contact time) increases [18] (Figure1A). In contrast, the vertical-flow perfusion path only comes in contact with cells along the height of the cell monolayer (in µm level). The perfusate flow can then be designed to reach and leave the cells across the culture area simultaneously, which in theory should create an evenly distributed media supply in the original concentration (Figure1B). There is also the mass transfer problem present in horizontal-flow (including cross-flow) reactors: the flow rate has to be carefully balanced to minimize shear stress and to optimize mass transfer, which in turn means that not all the perfusate will come into contact with the cells [19]. In the case of a vertical-flow reactor, the vertical flow pushes all the perfusate onto the cells, which maximizes the media supply to the cells with shear stress kept at a minimum. Moreover, the vertical-flow design’s culture area is easily scaled up because the perfusion is no longer made on 2D surfaces unlike many microfluidic chips [6,15,18,20]. All perfusion channels and tubing would be located above and below the culture chambers so that the footprint may be compact enough to accommodate multiple wells with sufficient well sizes, enabling the vertical-flow bioreactor design to be modular and scalable. Biosensors 2021, 11, 160 4 of 13 Biosensors 2021, 11, x FOR PEER REVIEW 4 of 13 Biosensors 2021, 11, x FOR PEER REVIEW 4 of 13

Perfusion In Perfusion In PerfusionPerfusion In In PerfusatePerfusate Out Out Flow enlarger Flow enlarger Membrane Filter Cap Membrane Filter Cap Flow distributor Flow distributor Culture chamber CultureCulture chamber membrane Culture membrane Flow distributor Bottom Flow distributor Bottom

Concentration Drain collection Concentration Flow Length Drain collection Flow Length Perfusate Out Perfusate Out

(A(A) ) (B(B) ) FigureFigureFigure 1. 1.1. Horizontal Horizontal-Horizontal- versus- versusversus v vertical-flow:erticalvertical-flow:-flow: (A (A) )Horizontal) HorizontalHorizontal flow flowflow brings bringsbrings an anan intrinsic intrinsicintrinsic gradient gradientgradient concentration concentrationconcentration when whenwhen there therethere is isis an anan absorptionabsorptionabsorption by byby the thethe cells cellscells along alongalong the thethe perfusion perfusionperfusion path. path.path. ( B ((BB) )Vertical) Vertical-flowVertical-flow-flow bioreactor bioreactorbioreactor distributes distributesdistributes the thethe perfusion perfusionperfusion evenly evenlyevenly across acrossacross the thethe culturecultureculture area, area,area, and and and make makes makes thesthe the perfusateperfusate perfusate reach reach reach the the the cells cells cells on on theon the the same same same plane plane plane simultaneously simultaneously simultaneously with with with the originalthe the original original media media media concentration. concen- concen- tration. (Illustration only, not draw on scale). tration.(Illustration (Illustration only, not only, draw not on draw scale). on scale).

ThisThis VfB VfB also also adopts adopts the the open open-capopen-cap-cap design design for for the the ease ease of of cell cell seeding seeding and and harvest, harvest, thereforethereforetherefore facilitating facilitatingfacilitating downstream downstream assays assays such such as as staining, staining, imaging, imaging, RNA RNA extraction, extraction, etc. etc. TheThe cap capcap of ofof VfB VfB VfB was was constructedconstructed constructed withwith with two two two layers layers layers of of micro-featuredof micro micro-featured-featured PMMA PMMA PMMA with with with a membrane a amem- mem- branefilterbrane filter sandwiched filter sandwiched sandwiched via permanent v iavia permanent permanent bonding, bonding, bonding, and the and and bottom the the bottom bottom consists consists consists of another of of another another two slayers two two slayersofslayers PMMA of of PMMA PMMA structure structure structure to form to to form the form culture the the culture culture chambers chambers chambers (Figures (Figure (Figure1B ands s1B 1B2 andF). and In2F) 2F) this. In. In setup,this this setup, setup, the thecellthe cell cell culture culture culture membranes membranes membranes can can becan reversiblybe be reversibly reversibly fixed fixed fixed into into individualinto individual individual culture culture culture chambers, chambers, chambers, via ringvia via ringclipsring clips clips (Figure (Figure (Figure3B). 3B). The 3B). The VfB The VfB was VfB was then was then fixedthen fixed fixed into into the into PMMAthe the PMMA PMMA jig, withjig, jig, with awith silicone a asilicone silicone ring ring toring seal to to sealtheseal the interfaces the interfaces interfaces between between between the rigidthe the rigid PMMArigid PMMA PMMA parts parts (Figureparts (Figure (Figure3A,C), 3A,C and3A,C), with ),and andtubings with with tubings tubings and valves and and valvesconnectedvalves connected connected to the to jig to the (Figurethe jig jig (Figure (Figure3D). 3D) 3D). .

(A(A) ) (B(B) )

(C(C) ) (D(D) )

Figure 2. Cont. BiosensorsBiosensors2021 2021,,11 11,, 160 x FOR PEER REVIEW 55 ofof 1313

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(E) (F) (E) (F) FigureFigure 2.2. Flow distributor optimization:optimization: ((AA)) SchematicSchematic designdesign ofof testingtesting chipchip whichwhich hashas samplesample collectioncollection channelschannels startingstarting alongalongFigure thethe 2. diameter Flowdiameter distributor and and was was optimization placed placed below below: ( theA the) distributorSchematic distributor design to collectto collect of thetesting the perfusate. perfusate. chip which (B) ( InB has) theIn sample the case case when collection when hole hole size channels size and positionsand starting posi- tions are evenly distributed on the flow distributor, perfusion velocity was shown to be higher through the central sample arealong evenly the distributeddiameter and on was the flowplaced distributor, below the perfusion distributor velocity to collect was the shown perfusate. to be higher(B) In throughthe case thewhen central hole samplesize and holes posi- holestions thanare evenly the periphery distributed ones. on ( theC) Simulation flow distributor, of flow perfusion velocity nearvelocity the wasexit ofshown the even to be-hole higher distributor through atthe the central overall sample flow than the periphery-1 ones. (C) Simulation of flow velocity near the exit of the even-hole distributor at the overall flow rate of rateholes of than 0.1 m theL hperiphery. (D) Dimension ones. (C )of Simulation finalized flowof flow distributor velocity neardesign. the ( exitE) Simulation of the even of-hole flow distributor velocity near at the the overall exit of flow the 0.1 mL h−1.(D) Dimension of finalized flow distributor design. (E) Simulation of flow velocity near the exit of the finalized finalizedrate of 0. 1flow mL distributor. h-1. (D) Dimension (F) Layer of st finalizedructure of flow the distributorVfB (double design. well design (E) Simulation as the prototype of flow describedvelocity near here). the exit of the flow distributor. (F) Layer structure of the VfB (double well design as the prototype described here). finalized flow distributor. (F) Layer structure of the VfB (double well design as the prototype described here). (A) (B) (A) (B)

Figure 3. Assembly of VfB: (A) The VfB components: left column: jig to encase VfB; middle column: VfB cap and bottom; right column: O-rings to seal jig-cap, O-rings to seal cap-bottom, ring FigureFigure 3.3.Assembly Assembly ofof VfB:VfB: ( A(A)) The The VfB VfB components: components: left left column: column: jig jig to to encase encase VfB; VfB; middle middle column: col- clips,umn: OVfB-ring cap to and seal bottom; bottom -rightjig. (B column:) Fix a cell O- ringsseeding to sealmembrane jig-cap, withO-rings a ring to sealclip. cap (C)- bottom,VfB encased ring VfB cap and bottom; right column: O-rings to seal jig-cap, O-rings to seal cap-bottom, ring clips, inclips, jig with O-ring screw to seal security. bottom (D-jig.) Connect (B) Fix perfusiona cell seeding tubing membrane to VfB. Scale with bara ring = 1 clip. cm. (C) VfB encased O-ring to seal bottom-jig. (B) Fix a cell seeding membrane with a ring clip. (C) VfB encased in jig in jig with screw security. (D) Connect perfusion tubing to VfB. Scale bar = 1 cm. with screwLayer security. 1 of the ( Dcap) Connect and Layer perfusion 2 of the tubing bottom to VfB. is Scalethe “flo barw = enlarger” 1 cm. , which interfaces with theLayer Φ1 1 mm of the (ID) cap perfusion and Layer tubing 2 of andthe bottomthe Φ15 is mm the membrane “flow enlarger”-filter., Thewhich entry interfaces orifice Layer 1 of the cap and Layer 2 of the bottom is the “flow enlarger”, which interfaces iswith a Φ1 the mm Φ1 through mm (ID) hole, perfusion linking tubing to an andinverted the Φ15 V shape mm membrane structure with-filter. 8 guidingThe entry grooves orifice with the Φ1 mm (ID) perfusion tubing and the Φ15 mm membrane-filter. The entry orifice on the inner wall, so that the flow could be easily wicking along the wall and fills the isis a aΦ Φ11 mmmm throughthrough hole,hole, linkinglinking toto anan invertedinverted VV shapeshape structurestructure withwith 88 guidingguiding grooves grooves whole shallow enlarger chamber (Figures 1B and 2F). onon the the inner inner wall, wall, so so that that the the flow flow could could be easily be easil wickingy wicking along along the wall the and wall fills and the fills whole the Layer 2 of the cap and Layer 1 of the bottom contains the “flow distributor” structure, shallowwhole shallow enlarger enlarger chamber chamber (Figures (1FigureB ands2 F).1B and 2F). which is an array of through holes with the diameters at 0.400, 0.480, 0.576, 0.691, 0.829, LayerLayer 2 2 ofof thethe capcap andand LayerLayer 11 ofof thethe bottombottom containscontains thethe “flow“flow distributor”distributor” structure,structure, 0.995 mm from center to periphery (Figure 2D). The numbers were optimized via CFD whichwhich isis anan arrayarray ofof throughthrough holesholes withwith thethe diametersdiameters atat 0.400,0.400, 0.480,0.480, 0.576,0.576, 0.691,0.691, 0.829,0.829, simulation in ANSYS (Figure 2E). 0.9950.995 mmmm fromfrom centercenter toto peripheryperiphery (Figure(Figure2 D).2D) The. The numbers numbers were were optimized optimized via via CFD CFD simulationsimulationIn our inconsiderationin ANSYSANSYS (Figure (Figure in coming2 E).2E). up with a good bioreactor design, we have summa- rizedIn Inthe our our ideal consideration consideration features of in cellin coming coming culture up up devices, with with a good a especially good bioreactor bioreactor bioreactors design, design, we capable have we have summarizedof perfusion summa- theculturerized ideal the and features ideal with features ofbiometic cell cultureof cellfeatures culture devices, mimicking devices, especially theespecially bioreactorsin vivo bioreactors microenvironment capable capable of perfusion ofof perfusioncells. culture We andhaveculture with then and biometic tried with to biometic featurescreate a mimickingVfBfeatures design mimicking theto meetin vivo all the microenvironmentthese in vivo requirements microenvironment of (Table cells. We1) .of have cells. then We triedhave tothen create tried a VfBto create design a VfB to meet design all theseto meet requirements all these requirements (Table1). (Table 1).

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Table 1. Considerations in VfB to meet with the ideal features of cell culture device and bioreactor.

Ideal Features of Cell Culture Device for Drug Testing Corresponding Considerations in VfB Design Biocompatible, nontoxic material [14] PMMA as main body, PET as cell culture substrate, with collagen coating Optically transparent [15] Transparent cell culture membrane for staining and imaging Large surface area for enough cells to attach [14] Cell culture chamber same as 24-well plate (Φ15 mm) Vertical-flow to ensure all media flow onto cells Efficient mass delivery [14] No ECM on top of cells to avoid additional adsorption of drug or media Easy to assemble, insert and retrieve culture parts [15] Open-cap design, allowing easy retrieval of cells and culture substrates Flexible configuration: allow co-culture [15] Allows for co-culture in mixture or different side of cell culture membrane Scalable or High throughput [15] Modular structure of culture wells and perfusion channels for easy scale-up Ideal Features of Perfusion bioreactor for drug testing Closed culture environment [16] Closed culture well during perfusion by embracing VfB inside the jig Leak proof [15] Seal rigid surfaces with silicone rings and fixed in jig via screws Allows use of different flow types/rates [15] Flow type/rates fully adjustable by syringe pump No air bubble formation [15] Simple structure without micro-channel features Even distribution of flow across the large culture area * Flow enlarger + Flow distributor to dissipate flow of media Low buffer volume for in-time media replenishment * Shallow culture chambers with vertical flow to efficiently replenish media Micro-pore membrane filter to filter shear stress Minimize the shear stress * Vertical flow enlarged from small to big cross section area (Φ1 to 15 mm) Biometic Features Maintain 3D cell shape as in vivo [14] Cells maintain 3D cuboidal shape with minimum stress fibers [11] Controllable Compaction * Cell compaction similar to intra-abdominal pressure (IAP) [11] * the unique features achieved by VfB.

Along with the abovementioned considerations in Table1, the key functions of a VfB are elaborated below. The cap would carry out the following functions: 1. Minimize the shear stress: At a given flow rate, the expansion of the cross section area perpendicular to the flow direction may result in the decrease of velocity, thus lowering the shear stress at the exit of the expansion structure, which serves as the “flow enlarger” in the VfB. The “membrane filter”, sandwiched between the two layers of the cap, also serves as a shear stress filter, as the membrane was track- etched with micro-pores (Φ0.4 or 3 µm) so that the high resistance of these pores will force the perfusion flow to sieve through the membrane. This would lower the impact of perfusion flow onto the fragile cells below the filter, mimicking the in vivo condition of plasma sieving through blood vessel walls (e.g., pore size Φ0.4 µm on mice vessels) [21] (Figures1B and3A). 2. Even distribution of flow across the whole cross section area: As the diameter of the perfusion tubing is as small as Φ1 mm while the diameter of the membrane filter is expanded to Φ15 mm, a “flow distributor” is needed to dissipate the flow to the periphery of the culture chamber and force the flow velocity to distribute evenly across the area of the culture chamber below (Figure2F). If the through hole array in the flow distributor were of the same size and arranged at the same intervals, the flow velocity of the media would be much faster at the center than at the sides (Figure2B) when taking sampling points across the diameter (Figure2A). It is therefore important to increase the flow resistance in the middle. The computational fluidic dynamic (CFD) simulation showed that the flow velocity was much more evenly distributed after this adjustment, other than the part for accommodating ring clips (Figure2C versus Figure2E). 3. Low buffer volume for in-time media replenishment: As the cells in the perfusion path of VfB receives 100% perfusate due to the vertical-flow design, the flow rate is controlled at a much lower level than the horizontal or cross-flow bioreactor types due to the minimized loss of flow portion in parallel with the cell seeding plane. The volume before the cell substrate, considering as the “buffer volume” was then kept at a low level so that the media replenishment rate may still be maintained. Since the cell culture area needs to be large, the height of the cell culture chamber, which is the distance between the membrane filter and the cell seeding membrane, needs to be Biosensors 2021, 11, 160 7 of 13

kept low. This is the reason why the culture chambers in VfB are shallow—to achieve a low buffer volume for in-time media replenishment. The bottom of the VfB functions as the cell culture module, with the PET membranes reversibly fixed into the culture wells as the cell seeding substrates. The features of the VfB culture module include: 1. Enough cell number per data point: In prevalent microfluidic culture devices, the cell number per data point is commonly limited by the micro-chambers. For example, the 3D-µFCCS (3D microfluidic channel-based cell culture system) previously developed could only accommodate several thousand hepatocytes [6], making it difficult to do downstream assays such as RNA extraction and immunofluorescence staining. Since the flow distributor may expand the area at the membrane filter part, the cell seeding membrane below should also be designed at the same area so as to accommodate enough cells for each chamber. 2. Controllable Compaction: In the report from Wang et al. [9], the compaction generated by the pressure applied on the ceiling of the elastic microchannel may positively modulate the cells inside. The compaction described in their paper, however, was performed by manually controlled tweezers. Here, the perfusion flow regulated by the external pump would also generate compaction force, but in a controllable way. When the flow resistance increases through integrating the micro-pore membrane at the outlet of the flow path, the pressure will build up until it is high enough to drive the perfusate through the nano pores. This pressure will then act as the compaction onto the cells cultured inside the chamber, in an even and controllable way. In our previous study, we have demonstrated that this compaction enhanced cell polarity and functions [11]. 3. Efficient mass delivery: The vertical-flow design allows all the media to pass through the cell culture membrane, enabling better media delivery to the cells. In order to supply enough O2 in the perfused media, a gas permeable tubing was used as the O2 generator before the perfusate reaches from syringe pump to the bioreactor. Specifically, in a drug testing application, it is important to avoid adsorption by the ECM (extra-cell matrix) overlay when adding the drug onto the cells in culture. Since the 2D cell culture setup in VfB may result in a similar morphology seen in 3D culture and in vivo, no overlay of ECM on top of the cells is needed, due to the compaction culture condition [11]. Additional features of a VfB would include: 1. Biocompatibility: All wet parts which comes into contact with culture media or cells were made from biocompatible materials. The main body is constructed with thermoplastic polymethyl methacrylate (PMMA), which has been reported to be compatible with various cell types including hepatocytes [22,23]. The VfB adopted commercially available Transwell®Polyester (PET) membranes as the cell seeding substrates, which are proven to be cell friendly and used widely for cell culture. 2. Scalable Throughput: For drug testing purposes, a multi-well design is preferred for the purpose of high throughput. The VfB shares the same design culture chamber unit as a module, which enables flexible throughput from a single well, to double, 24 wells or higher. For instance, in order to scale up to a 24-well culture plate footprint, we could fabricate 6 groups of 4-well units to combine as an array [11]. The VfB is therefore flexible in throughput, and able to cater to various scale requirements in product design.

3.2. Device Fabrication and Thermo-Bonding Optimization The main body of VfB was fabricated from PMMA plates machined according to the designed features (Figure2F). As shown in Figure1B, there were two bonding pro- cesses required for each VfB: two layers of PMMA sandwiched with PET membranes as the cap, and another two layers thermo-bonded without membranes as the bottom. As Biosensors 2021, 11, x FOR PEER REVIEW 8 of 13 Biosensors 2021, 11, 160 8 of 13

and another two layers thermo-bonded without membranes as the bottom. As PMMA wasPMMA proven was to proven be a biocompatible to be a biocompatible thermoplasti thermoplastic,c, it is advantageous it is advantageous to use thermo-bond- to use thermo- ingbonding as it does as it not does involve not involve adhesives adhesives or tapes, or tapes, lowering lowering the chances the chances of complications of complications from introducingfrom introducing foreign foreign materials, materials, such as such micro- asfeature micro-feature blockages, blockages, surface property, surface property, and bi- ocompatibilityand biocompatibility changes, changes, etc. [24]. etc. In the [24 ].late Inr stage the later of scaled-up stage of scaled-upproduction, production, the simplicity the andsimplicity reliability and of reliability thermo-bonding of thermo-bonding would also would enable also the enable low cost the lowand cost high and yield high manu- yield facturingmanufacturing of the ofmicrofluidic the microfluidic device device [23]. [23]. InIn this this paper, paper, we we have have tested tested two two methods methods of of thermo-bonding thermo-bonding to to assemble assemble the the VfB. VfB. TheThe first first one one involved involved applying applying high high pressure pressure (as (as high high as as 200 psi) on PMMA via metal clampingclamping plates plates and and pressurized pressurized hotplates hotplates (Figure (Figure 44A).A). The bonding surfaces are required toto be be plasma plasma treated treated after after wiping wiping down down with with isopropyl isopropyl alcohol alcohol (IPA) (IPA) and and N2-blow N2-blow drying. drying. ◦ AsAs the the glass glass transition transition point point (Tg) (Tg) for PMMA PMMA is 105 °CC[ [25],25], the the temperature temperature of of the the heating heating ◦ plateplate was was adjusted adjusted from from 93 93 to to 96 °C,C, and and the the bonding time time was was 20–40 20–40 min based on the observationobservation and and adjustment adjustment for for the the different different batches of PMMA materials. The The processing processing timetime was was short, but therethere werewere limitationslimitations on on the the bonding bonding area area and and evenness, evenness, and and the the use use of ofhigh high pressure pressure in bondingin bonding the the VfB VfB meant meant that th theat accuratethe accurate temperature temperature control control was criticalwas crit- to icalavoid to over-avoidor over- under-bonding. or under-bonding. Optimizations Optimizations were needed were needed for each for batch, each and batch, the bondingand the effect was highly dependent on the operator’s skill. When under-bonding occurs, the parts bonding effect was highly dependent on the operator’s skill. When under-bonding occurs, may still be salvaged by increasing the initial 15-min bonding time by 10–15 min at 1–2 ◦C the parts may still be salvaged by increasing the initial 15-min bonding time by 10–15 min higher than the initial temperature set at 95 ◦C. When over-bonding occurs, the collapse at 1–2 °C higher than the initial temperature set at 95 °C. When over-bonding occurs, the of micro-features and the blocking of the shallow channels and chambers may largely collapse of micro-features and the blocking of the shallow channels and chambers may change the flow resistance of the filters, causing the whole part to become unusable. The largely change the flow resistance of the filters, causing the whole part to become unusa- whole process involves much estimation and trial-and-error, and was heavily dependent ble. The whole process involves much estimation and trial-and-error, and was heavily on manual operation and observation through the naked eye. This method is therefore dependent on manual operation and observation through the naked eye. This method is only suitable for rapid prototyping. therefore only suitable for rapid prototyping.

(A) (B) (C)

120 ºC, 1h (D)

Ramping 1h

60 ºC, 1h Room Room Temp Temp

(E) (F)

FigureFigure 4. 4. Thermo-bondingThermo-bonding of VfB: ( A) Thermo-bonding machinemachine byby hydraulichydraulic pressure.pressure. ( B(B)) Vacuum Vac- uumoven oven for surface for surface dehydration. dehydration. (C) (C High) High temperature temperature oven oven for for thermo-bonding. thermo-bonding. ((DD)) OptimizedOpti- mized bonding protocol in high temperature oven. (E,F) The finishing surface of thermo-bonded bonding protocol in high temperature oven. (E,F) The ﬁnishing surface of thermo-bonded VfB with VfB with (E) and without (F) overnight vacuum oven dehydration. Scale bars = 1 cm. (E) and without (F) overnight vacuum oven dehydration. Scale bars = 1 cm.

AnAn improved improved method method used used was was to to gently gently apply apply pressure pressure (via (via paper paper clips clips onto onto non- non- bondingbonding clamping clamping plates plates like like thick thick glass) glass) onto onto the the PMMA PMMA layers layers and put the bonding partsparts into into a a high high temperature temperature oven oven to to allow allow bonding bonding at 120 120 °C◦C (Figure (Figure 44C).C). A ready-to-use Biosensors 2021, 11, 160 9 of 13

temperature protocol is shown in Figure4D. As the bonding temperature is above the Tg of PMMA at 105 ◦C, the flowing of the material at the interface makes the bonding strength strong enough to hold the sandwiched PET membrane, even though PMMA and PET do not bond with each other. Besides, the space of a high-temperature oven allows for a much bigger bonding area and higher throughput than pressurized hotplates. The finished products were perfectly transparent with no visible delamination or defects (Figure4E). Notably, the oven bonding required the bonding surface to be vacuum dried at 80 ◦C overnight (Figure4B). If not done, the evaporation of adsorbed moisture may result in an uneven finish, with bubbles trapped on the bonded surface (Figure4F). This mode of production is ideal for small scale production when the design and fabrication protocols are confirmed.

3.3. Prepare VfB for Cell Seeding The VfB was connected with PEET tubing and four-way valves (Upchurch Scientific, IDEX Corporation, Lake Forest, IL, USA) through a machined PMMA jig. Silicone O-rings were used to couple and seal the hard surfaces between the VfB and jig, so that the perfusate can be delivered from a syringe to VfB. Notably, there is a silicone air-permeable-water- proof tubing (Silastic® Laboratory tubing, 7-5224 Cat#508-009, Cole-Parmer, Vernon Hills, IL, USA) in the perfusion path, which serves as the O2/CO2 exchanger when the perfusion culture was done inside the CO2 incubator. The abovementioned parts were then prepared for the cell seeding procedures as shown in Figure3. In order to sterilize VfB for mammalian cell culture, the tubing, valves, and other PEET parts were autoclaved at 105 ◦C for 30 min and kept wet until used. Other bioreactor parts made from PMMA and silicone should not be autoclaved, but rather soaked in 70% ethanol overnight and then air dried on autoclaved tissue inside a UV-sterilized biosafety cabinet (BSC). On the day of cell seeding, the sterilized flow divider and culture layers were assembled inside the BSC, in the jig connected with perfusion tubing and with silicone O-rings used as water-proof sealing (Figure3C). The sterilized VfB with perfusion path needs to be primed before seeding. First, the whole flow path needs to be rinsed with a continuous perfusion of phosphate buffer saline (PBS) in the volume of at least 3 mL/well. This is to remove leftover water or ethanol from the sterilization steps, as these are hazardous to hepatocytes [26]. The PBS was then replaced by 0.2% bovine serum albumin (BSA)-PBS solution for 1 mL/well and left in the flow path with valves closed for >1 h. Immediate before cell seeding, the BSA-PBS solution was replaced with fresh William E medium for rat hepatocytes (Rat-Hep-WE). The VfB was then ready to accept the cell seeding membranes. The PET Transwell® membrane with the track-etched 0.4 µm pore membrane (Corning 3450) was chosen as the cell seeding membranes for its transparency. When taken off inside the BSC with sterilized blades, the membrane was kept larger than 16 mm so as to leave enough space for fixing onto the VfB. Coating of the membrane with extracellular matrix (ECM) facilitates the cell attachment. Here, we used the acidic collagen coating method with the briefed steps as below: 1. Prepare 1:10 dilution of collagen gel stock in 0.1 N HCl at room temperature to achieve 0.3 mg/mL acidic collagen. 2. Soak PET membrane in acidic collagen with a volume of 1 mL/well in 12-well plate. Make sure the whole cell seeding areas are immersed. 3. Keep the 12-well plate in 37 ◦C incubator for 2 to 3 h. 4. Wash three times with 5 min interval in 2 mL/well PBS in order to remove acidic solution thoroughly. 5. Replace PBS with 1 mL/well Rat-Hep-WE and keep the membrane wet until prior to cell seeding. The coating needs to be freshly done before cell seeding, and the coated membranes fixed onto the primed cell culture wells with sterile ring clips inside the BSC so as to get the VfB ready for cell seeding (Figure3B). After seeding was completed, the cap of Biosensors 2021, 11, x FOR PEER REVIEW 10 of 13

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the VfB ready for cell seeding (Figure 3B). After seeding was completed, the cap of VfB VfBwas was closed closed immediately; immediately; then then the whole the whole VfB VfBwas wasencased encased in the in jig the and jig andconnected connected with perfusion tubings and syringes (Figure 3C,D), and moved to a CO2 incubator for subse- with perfusion tubings and syringes (Figure3C,D), and moved to a CO 2 incubator for subsequentquent culture. culture.

3.4.3.4. OptimizationOptimization of of Cell Cell Seeding Seeding Density Density in in the the VfB VfB DuringDuringin in vitro vitroculture, culture, the the hepatic hepatic functions functions and and cell cell number number of of hepatocytes hepatocytes tend tend to to dropdrop when when the the culture culture time time was was extended, extended, especially especially in in perfusion perfusion cultures, cultures, where where the the risk risk ofof cell cell loss loss is is higher higher than than that that in in static static cultures cultures due due to to the the introduction introduction of of the the culture culture media media ﬂowflow [ 27[27].]. Bioreactor Bioreactor designers designers tend tend to to increase increase the the density density of of cell cell seeding seeding to to compensate compensate forfor hepatic hepatic function function and and cell cell loss. loss. However, However, we we believe believe that that the the optimum optimum density density should should bebe optimized optimized against against hepatic hepatic functions, functions, as as unnecessary unnecessary high high seeding seeding density density does does not not only only increaseincrease the the cost cost of of tests tests but but may may also also incur incur negative negative impact impact on on cell cell performance. performance. Since Since ureaurea production production is is an an important important hepatic hepatic function, function, which which is is easily easily quantiﬁed quantified by by sampling sampling thethe perfusate perfusate on on a a daily daily basis basis without without affecting affecting the the culture culture process, process, we we used used it it to to optimize optimize thethe cell cell seeding seeding density density in in each each well well of of the the VfB VfB (Figure (Figure5). 5).

FigureFigure 5. 5.Cell Cell seeding seeding density density optimization: optimization: Urea Urea production production was was tested tested for fo 4r days 4 days for for three three different differ- seedingent seeding densities: densities: 0.2, 0.4, 0.2, and 0.4, 0.8 and million 0.8 million cells/well, cells/well, in both in compactionboth compaction (in VfB) (in and VfB) in and static in culturestatic culture (in 24-well culture plate). All quantification results normalized to seeded cell numbers. (in 24-well culture plate). All quantiﬁcation results normalized to seeded cell numbers. Error bars Error bars show the standard error of the mean (SEM, n = 2–4). Scale bars = 50 μm. show the standard error of the mean (SEM, n = 2–4). Scale bars = 50 µm.

TheThe hepatocytes hepatocytes cultured cultured in in the the VfB VfB withwith differentdifferent seeding seeding densitiesdensities werewere compared,compared, togethertogether with with the the static static control control which which was was seeded seeded on on the the same same type type of of PET PET membrane membrane in in aa monolayer monolayer andand cultured cultured in in a a 24-well 24-well culture culture plate. plate. As As shown shown in in Figure Figure5 ,5, hepatocytes hepatocytes formedformed coloniescolonies inin allall the the cultural cultural conditionsconditions wewe tested,tested, withwith differentdifferent morphologymorphology ob-ob- servedserved at at the the end end of of the the 4-day 4-day culture: culture: for for seeding seeding density density at at 0.2 0.2 and and 0.4 0.4 million million cells/well, cells/well, aa monolayer monolayer of of colonies colonies appeared appeared in in both both static static and and compaction compaction culture; culture; while while 0.8 0.8 million million cells/wellcells/well inin compactioncompaction cultureculture showedshowed severesevere clumpingclumping ofof cellscells on on the the membrane membrane with with mixedmixed cell cell morphologies, morphologies, andand thosethose inin static static culture culture showed showed peeling peeling off off of of cells cells from from the the membranesmembranes during during the the daily daily media media change change (picture (picture not not shown). shown). When When the the hepatic hepatic function func- oftion urea of productionurea production was quantiﬁed was quantified daily, alldaily, compaction all compaction cultured cultured hepatocytes hepatocytes produced pro- higherduced ureahigher than urea those than in those static culture,in static whileculture, the while 0.2 million the 0.2 cells/well million cells/well showed theshowed highest the ureahighest production urea production rate. Therefore, rate. Therefore, the seeding the density seeding of density 0.2 million of 0.2 cells/well million wascells regarded/well was asregarded the optimized as the optimized condition andcondition was used and for was the used following for the studies.following studies.

3.5.3.5. HepaticHepatic Functions Functions Were Were Enhanced Enhanced to to Facilitate Facilitate Drug Drug Testing Testing Applications Applications ForFor drug drug testing testing applications, applications, it isitpreferable is preferable to maintain to maintain cellfunctions cell functions in the in sustained the sus- culturetained soculture that theso sub-chronicthat the sub- orchronic chronic or effect chronic of drugseffect couldof drugs be tested.could be In ourtested. previous In our work, the compaction culture was already shown to preserve the hepatocytes’ morphology Biosensors 2021, 11, x FOR PEER REVIEW 11 of 13

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previous work, the compaction culture was already shown to preserve the hepatocytes’ aftermorphology 14 days after [11]. 14 In days this paper,[11]. In we this have paper, observed we have that observed the urea that and the CYP urea functions and CYP of hepatocytesfunctions of culturedhepatocytes in the cultured VfB could in the be maintainedVfB could be and maintained enhanced and for up enhanced to 14 days, for usingup to the14 days, static using culture the as static a control culture (Figure as a 6control). (Figure 6).

FigureFigure 6.6. Hepatic function assays targetingtargeting drugdrug testingtesting application:application: (A)) UreaUrea productionproduction overover 22 weeks.weeks. ((BB––DD)) BasicBasic CYPCYP level tested via LC-MC. Cells were harvested after 14-day culture: (B) CYP1A2. (C) CYP2B2. (D) CYP3A2. level tested via LC-MC. Cells were harvested after 14-day culture: (B) CYP1A2. (C) CYP2B2. (D) CYP3A2.

For urea production, the hepatocytes in VfB maintained at 8 to 2020 foldsfolds higherhigher thanthan the static static culture culture throughout throughout the the whole whole 2-week 2-week culture culture period period (Figure 6 (FigureA). For 6A)the CYP1A2,. For the 2B2CYP1A2, and 3A2 2B2 resultsand 3A2 of theresults cells of harvested the cells harvested at the end at of the the end 2-week of the culture, 2-week the culture, perfusion the culturesperfusion all cultures exhibited all higher exhibited production higher levelsproduction than the levels static than control the asstatic well control (Figure as6B–D). well All(Figure thequantification 6B–D). All the results quantification here show results that thehere VfB show enhanced that the the VfB hepatic enhanced functions the hepatic of the hepatocytesfunctions of forthe ahepatocytes long-term culture,for a long which-term may culture, be a which promising may drugbe a promising testing platform. drug testing platform. 4. Conclusions 4. ConclusionsBioreactors for in vitro cell culture are useful tools for both scientific research (e.g., diseaseBioreactors modeling, for tissue in vitro engineering) cell culture and are clinical useful applicationstools for both (e.g., scientific artificial research organs, (e.g. cell, therapy,disease modeling, drug testing) tissue [1– engineering)3,28–30]. An idealand clinical bioreactor applications should abide (e.g., by artificial certain organs, guidelines cell totherapy, provide drug a suitable testing) microenvironment [1–3,28–30]. An ideal for cellsbioreactor to maintain should their abide viability by certain and guidelines functions throughoutto provide a thesuitable culture microenvironment period. This includes for cells ensuring to maintain efficient their mass viability delivery and to functions support athroughout sufficient numberthe culture of cellsperiod. growing This includes inside theensuring bioreactor, efficient providing mass delivery in-time to media support replenishment,a sufficient number supplied of evenlycells growing to all the inside cells, the mimicking bioreactor, the providing environment in- intime the media body forre- 3Dplenishment, and compaction supplied culture, evenly etc. to all [11 ].the In cells, conceptualizing mimicking the and environment designing bioreactors in the body as for a product3D and forcompaction future industrialization, culture, etc. [11] the. In scalability, conceptualizing manufacturability, and designing biocompatibility, bioreactors andas a easeproduct of sample for future retrieval industrialization, are all important the scalability, considerations. manufacturability, Since our previously biocompatibility, published vertical-flowand ease of sample compaction retrieval bioreactor are all arrayimportant (VCBA) considerations. has been proven Since to our meet previously the abovemen- pub- tionedlished guidelinesvertical-flow [11 compaction], we seek to bioreactor elaborate on array the methodology(VCBA) has been and principles proven to of meet design, the setup,abovem optimization,entioned guidelines and test [11] of the, we vertical-flow seek to elaborate bioreactor on the (VfB) methodology in this paper, and principles using the double-wellof design, setup, VfB asoptimization, a case study. and test of the vertical-flow bioreactor (VfB) in this paper, usingIn the order double to meet-well theVfB requirements as a case study. of an ideal bioreactor, the design principles were carefullyIn order established, to meet suchthe requirements as the vertical-flow of an ideal perfusion bioreactor, to ensure the design mass transfer, principles the were flow enlargercarefully andestablished, distributor such to as expand the vertical the effective-flow perfusion cell culture to area,ensure the mass modular transfer, culture the flow unit design for easy scale-up, etc. The components were carefully chosen, such as the micro- enlarger and distributor to expand the effective cell culture area, the modular culture unit pore membrane as the shear stress membrane to protect the fragile hepatocytes cultured design for easy scale-up, etc. The components were carefully chosen, such as the micro- underneath, the biocompatible thermoplastic PMMA as the main construction material, the pore membrane as the shear stress membrane to protect the fragile hepatocytes cultured transparent cell culture membrane with reversible assembly to allow convenient retrieval Biosensors 2021, 11, 160 12 of 13

for downstream assays such as immunostaining, LC/MS, cell lysis, and PCR, etc. The fabrication process was discussed, with an elaboration on the thermo-bonding process optimization to form a strong sandwich structure with PET membrane ﬁlter to bear the elevated ﬂow pressure generated inside the culture chamber, which was then used as the compaction force for hepatocyte culture. The bioreactor prototype proved to enhance the hepatic functions of the cultured hepatocytes over sustained culture and showed promise in commercialization with scalable throughput as a manufacturable product.

Author Contributions: Conceptualization, L.Z. and H.Y.; methodology, L.Z., Z.W. and H.X.; software, H.X.; validation, L.Z.; formal analysis, L.Z.; investigation, L.Z.; resources, L.Z., Z.W. and H.Y.; data curation, L.Z. and H.X.; writing—original draft preparation, L.Z.; writing—review and editing, Z.W.; visualization, L.Z.; supervision, H.Y.; project administration, Z.W. and H.Y.; funding acquisition, H.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by the Institute of Bioengineering and Nanotechnol- ogy (IBN), Singapore Institute of Manufacturing Technology (SIMTech), National Medical Research Council (NMRC), JCO-DP, A*STAR, EMULSION & IFCS, Ministry of Education, SMART CAMP and Mechanobiology Institute (MBI) of Singapore funding to HYU. Liang Zhu was an MBI research scholar. Institutional Review Board Statement: The animals used in the experiments were handled under the approval of Institutional Animal Care and Use Committee (IACUC) of National University of Singapore (NUS). No human samples or participants were used, and no other ethics approval is required. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in https:// scholarbank.nus.edu.sg/handle/10635/124178 (accessed on 1 May 2021). Acknowledgments: We thank Yi Chin Toh, Jiangwa Xing, Abhishek Ananthanarayanan, Lei Xia, and Daphne Yen Peng Seah, Yinghua Qu, Ng Chan Wei and Fang Yu for technical support and scientific discussions. We also thank Evan Liao Sheng Yang for editing and proof reading. Conflicts of Interest: The authors declare no conflict of interest.

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