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BMES High School Student Chapter Member

HS Poster

Poster 77 - Fabrication and Validation of Miniature Perfused Channel Bioreactors

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: A hollow fiber bioreactor (HFB) is a 4-port device that has a variety of biotechnology applications. It consists of ports that connect to the inlet and outlet of HFs and Extra Capillary Space (ECS), the region outside of the fibers. HFBs enhance solute transport between Intra-Capillary Space (ICS) and ECS. HFBs are used for generating cell products,[6] with use growing ~16% annually.[8]

The ECS provides a region in which cells can grow and elaborate matrix. Agarose hydrogels can encapsulate cells, such as chondrocyes, in a 3-dimensional environment.[1,5,7,12] Agarose could be used to encapsulate cells in the ECS of HFBs. For tissue engineering studies, small volume HFBs would be useful but few (ECS of ~4 mL) are available.[2] HFBs can be fabricated by manually gluing HFs between inlet and outlets.[10]

The fibers of HFB provide not only channels for fluid flow but also a semi-permeable membrane to govern ICS-ECS transport. Once solutes have passed through the membrane to the ECS, diffusion may govern transport, with a time constant proportional to distance squared.[3] HFB transport are assessed by solute kinetics.[13]

My study aimed to develop and validate a method for creating Perfused Channel Bioreactors (PCBs), like HFBs with variable channels, but without membrane. The specific aims were to (1) develop a process for creating small PCBs of variable channel density, and (2) determine if transport in PCBs to the ECS was governed by L, the channel gap.

Materials and

Methods: Aim 1. PCB were fabricated from 12.7mm long polycarbonate tubes. Holes were drilled into one side to create ECS ports. Endcaps were 3D-printed with either 1 or 7 holes for channels, and included O-rings for sealing. PCB were assembled by threading stainless steel wires through endcap holes, injecting molten (40C) 2% agarose into ECS through ports, cooling to gel, removing wires, attaching tubing to endcaps, and plugging the side ports.

Aim 2. The effect of fiber density, affecting diffusion distance, L, on solute transport in PCB was assessed.
Experimental groups were: (G1 CTRL) agarose cylinder of various lengths (2L), L=6.4, 9.6, and 12.7mm, without dye; (G2) similar cylinders, immersed in food dye; (G3) L=1 mm rectangular blocks (2mm wide, 10mm length) immersed in dye; (G4, 1-fiber PCB) and (G5, 7-fiber PCB) of 0.25 mL volume, with dye pumped through the channels. Working solution was created from green food dye diluted to 100x with water.
Transport trials were conducted on samples (n=3/group). Photos were taken of cylinders, blocks, and PCBs initially and after various times (2, 5, 10, 15, 20, 30 min; 1, 6, 12, 24, 48 hr). Images were imported into ImageJ, and analyzed for gray-scale intensity in the central region of the cylinder, block, and PCB. Data are described as mean and SD, with ANOVA and planned comparisons in progress.

Results, Conclusions, and Discussions: Aim 1. To compare effects of fiber density (Figure 1), 1-channel and 7-channel PCBs were successfully fabricated (Fig.2A-C). After dye perfusion, compared to the initial water-perfused state (Figure 2D,G), dye was visible in the channels (Fig.2E-H) and surrounding agarose-filled ECS. PCBs were fluid-tight.
Aim 2. Just after exposure to dye (G2-G5, t=0+), dye was evident in regions adjacent to the source, but not distant regions. In G2 cylinders, dye penetrated the ends (Fig.3A-C.inset.i, top/bottom); in G3 blocks, dye penetrated the sides (Fig.3F.inset.i), with corresponding intensity profiles. G4-G5 PCBs showed dye in channels (Fig.3D,E.inset.i), but little in ECS, with intensity profiles showing 1 and 3 troughs, respectively, the latter coinciding with overlapping 2/3/2 channels.
Dye gradually (Fig.3.insets.ii-iv) reached steady levels throughout (Fig.3.inset.v). The time course (gray-scale value, Fig.4) varied with sample geometry. The time to half-maximal change, T1/2, was 6, 12, 18 hr, for L=6.4, 9.5, 12.7 mm cylinders. The T1/2 for the Group 3 1-channel PCB, L=3.2 mm, was ~30 min, while that for Group 4 7-channel PCB, L=1.0 mm, was < 5 min, similar to that for the L=1 mm block.
Thus, a miniature PCB was developed. Such PCB could be used for engineering tissues with hydrogel-encapsulating cells in the ECS. In addition, material or biological membranes could be incorporated at the ICS-ECS boundary.

Acknowledgements (Optional):

References (Optional): [1] Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. 10:745-58. 1992. doi: 10.1002/jor.1100100602.
[2] Cartridges. FiberCell Systems. https://www.fibercellsystems.com/products/cartridges/
[3] Crank J. The Mathematics of Diffusion, Second Ed. 1979. Oxford University Press.
[4] Eghbali H, Nava MM, Mohebbi-Kalhori D, Raimondi MT. Hollow fiber bioreactor technology for tissue engineering applications. Int J Artif Organs. 39: 1-15, 2016. https://doi.org/10.5301/jiao.5000466
[5] Hampton research https://hamptonresearch.com/uploads/support_materials/HR8-092_UserGuide.pdf
[6] Hopkinson, J. Hollow fiber cell culture systems for economical cell-product manufacturing. Nat Biotechnol, 3:225–30, 1985. doi:10.1038/nbt0385-225
[7] Hou Y, Mi K, Sun L, Zhou K, Wang L, Zhang L, Liu Z, Huang L. The application of hollow fiber cartridge in biomedicine. Pharmaceutics. 14:1485, 2022. doi: 10.3390/pharmaceutics14071485.
[8] Iwamoto M, Sato K, Nakashima K, Fuchihata H, Suzuki F, Kato Y. Regulation of colony formation of differentiated chondrocytes in soft agar by transforming growth factor-beta. Biochem Biophys Res Commun. 159:1006-11, 1989. doi: 10.1016/0006-291x(89)92208-0.
[9] Knazek RA, Gullino PM, Kohler PO, Dedrick RL. Cell culture on artificial capillaries: an approach to tissue growth in vitro. Science. 178:65-6, 1972. doi: 10.1126/science.178.4056.65.
[10] Kucharska AM, Kuettner KE, Kimura JH. Biochemical characterization of long-term culture of the Swarm rat chondrosarcoma chondrocytes in agarose. J Orthop Res. 8:781-92, 1990. doi: 10.1002/jor.1100080602.
[11] Kumar S, Wittmann C, Heinzle E. Minibioreactors. Biotechnol Lett. 26:1-10, 2004. doi: 10.1023/b:bile.0000009469.69116.03.
[12] Single-use Bioreactors Market Size & Share Report. 2030. https://www.grandviewresearch.com/industry-analysis/single-use-bioreactors-market-report
[13] Shipley RJ, Davidson AJ, Chan K, Chaudhuri JB, Waters SL, Ellis MJ. A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors. Biotech Bioeng. 108: 1450-61, 2011. doi: 10.1002/bit.23062
[14] Storm MP, Sorrell I, Shipley R, Regan S, Luetchford KA, Sathish J, Webb S, Ellis MJ. Hollow fiber bioreactors for in vivo-like mammalian tissue culture. J Vis Exp. 26:53431, 2016. doi: 10.3791/53431