Biomedical Microdevices

, Volume 10, Issue 1, pp 117–121 | Cite as

Microfluidic environment for high density hepatocyte culture

  • Mimi Y. Zhang
  • Philip J. Lee
  • Paul J. Hung
  • Terry Johnson
  • Luke P. Lee
  • Mohammed R. K. Mofrad
Article

Abstract

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm2) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 μl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.

Keywords

Microfluidics Cell culture Hepatocytes Bioreactor 

References

  1. D.R. Albrecht, G.H. Underhill, T.B. Wassermann, R.L. Sah, S.N. Bhatia, Probing the role of multicellular organization in three-dimensional microenvironments. Nat. Methods 3(5), 369–375 (2006)CrossRefGoogle Scholar
  2. J.W. Allen, S.N. Bhatia, Improving the next generation of bioartificial liver devices. Semin. Cell Dev. Biol. 13(6), 447–454 (2002)CrossRefGoogle Scholar
  3. L. De Bartolo, G. Jarosch-Von Schweder, A. Haverich, A. Bader, A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions. Biotechnol. Prog. 16(1), 102–108 (2000)CrossRefGoogle Scholar
  4. T. Elkayam, S. Amitay-Shaprut, M. Dvir-Ginzberg, T. Harel, S. Cohen, Enhancing the drug metabolism activities of C3A—a human hepatocyte cell line—by tissue engineering within alginate scaffolds. Tissue Eng. 12(5), 1357–1368 (2006)CrossRefGoogle Scholar
  5. S. Enosawa, T. Miyashita, X.K. Li, S. Suzuki, H. Amemiya, T. Omasa, K. Suga, T. Matsumura, A bioreactor with glutamine synthetase-transfected recombinant HepG2 cells exhibits ammonia removal activity without the need for added cofactors and substrates: Advantage of a cellular bioreactor over enzyme-immobilized beads. Journal of Artificial Organs 4(4), 348–352 (2001)CrossRefGoogle Scholar
  6. J. Fukuda, K. Nakazawa, Orderly arrangement of hepatocyte spheroids on a microfabricated chip. Tissue Eng. 11(7–8), 1254–1262 (2005)CrossRefGoogle Scholar
  7. J. Fukuda, K. Okamura, K. Nakazawa, H. Ijima, Y. Yamashita, M. Shimada, K. Shirabe, E. Tsujita, K. Sugimachi, K. Funatsu, Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2). Cell Transplant. 12(1), 51–58 (2003)Google Scholar
  8. J. Fukuda, K. Okamura, K. Ishihara, H. Mizumoto, K. Nakazawa, H. Ijima, T. Kajiwara, K. Funatsu, Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver. Cell Transplant. 14(10), 819–827 (2005)Google Scholar
  9. C. Guguen-Guillouzo, M. Bourel, A. Guillouzo, Human hepatocyte cultures. Prog. Liver Dis. 8, 33–50 (1986)Google Scholar
  10. E. Heinicke, U. Kumar, D.G. Munoz, Quantitative dot-blot assay for proteins using enhanced chemiluminescence. J. Immunol. Methods 152(2), 227–236 (1992)CrossRefGoogle Scholar
  11. B.B. Knowles, C.C. Howe, D.P. Aden, Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209(4455), 497–499 (1980)CrossRefGoogle Scholar
  12. E. Leclerc, Y. Sakai, T. Fujii, Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes. Biotechnol. Prog. 20(3), 750–755 (2004)CrossRefGoogle Scholar
  13. H.L. Lee, P. Boccazzi, R.J. Ram, A.J. Sinskey, Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control. Lab. Chip. 6(9), 1229–1235 (2006a)CrossRefGoogle Scholar
  14. P.J. Lee, P.J. Hung, V.M. Rao, L.P. Lee, Nanoliter scale microbioreactor array for quantitative cell biology. Biotechnol. Bioeng. 94(1), 5–14 (2006b)CrossRefGoogle Scholar
  15. P.J. Lee, P.J. Hung, L.P. Lee, An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture. Biotechnol. Bioeng. 97(5), 1340–1346 (2007)CrossRefGoogle Scholar
  16. K. Nakazawa, S.W. Lee, J. Fukuda, D.H. Yang, T. Kunitake, Hepatocyte spheroid formation on a titanium dioxide gel surface and hepatocyte long-term culture. J. Mater. Sci. Mater. Med. 17(4), 359–364 (2006)CrossRefGoogle Scholar
  17. S.L. Nyberg, J. Hardin, B. Amiot, U.A. Argikar, R.P. Remmel, P. Rinaldo, Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver. Liver Transplant. 11(8), 901–910 (2005)CrossRefGoogle Scholar
  18. S. Ostrovidov, J. Jiang, Y. Sakai, T. Fujii, Membrane-based PDMS microbioreactor for perfused 3D primary rat hepatocyte cultures. Biomed. Microdevices 6(4), 279–287 (2004)CrossRefGoogle Scholar
  19. J.K. Park, D.H. Lee, Bioartificial liver systems: current status and future perspective. J. Biosci. Bioeng. 99(4), 311–319 (2005)CrossRefGoogle Scholar
  20. M.J. Powers, K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A. Capitano, A. Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R. Kamm, L.G. Griffith, A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 78(3), 257–269 (2002)CrossRefGoogle Scholar
  21. S.K. Sia, G.M. Whitesides, Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24(21), 3563–3576 (2003)CrossRefGoogle Scholar
  22. A.J. Strain, J.M. Neuberger, A bioartificial liver—state of the art. Science 295(5557), 1005–1009 (2002)CrossRefGoogle Scholar
  23. E. Torok, J.M. Pollok, P.X. Ma, P.M. Kaufmann, M. Dandri, J. Petersen, M.R. Burda, D. Kluth, F. Perner, X. Rogiers, Optimization of hepatocyte spheroid formation for hepatic tissue engineering on three-dimensional biodegradable polymer within a flow bioreactor prior to implantation. Cells Tissues Organs 169(1), 34–41 (2001)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Mimi Y. Zhang
    • 2
  • Philip J. Lee
    • 1
  • Paul J. Hung
    • 1
  • Terry Johnson
    • 2
  • Luke P. Lee
    • 2
  • Mohammed R. K. Mofrad
    • 2
  1. 1.CellASIC CorporationSan LeandroUSA
  2. 2.UCSF/UC Berkeley Joint Graduate Group in BioengineeringBerkeleyUSA

Personalised recommendations