Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System

  • Ce Gu
  • Miaomiao Chai
  • Jiaxing Liu
  • Hui Wang
  • Wenjing Du
  • Yan ZhouEmail author
  • Wen-Song Tan
Original Article


Background and Aims

Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article.


hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting.


During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-β1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers.


Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.


Transdifferentiated hiHeps BAL Serum-free medium Microcarrier culture Fibronectin 



We thank the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences for providing hiHeps and the cell culturing protocol. Ce Gu performed all the experiments and wrote the manuscript. Miaomiao Chai, Jiaxing Liu, Hui Wang, and Wenjing Du were involved in useful discussions during the development of this study. Yan Zhou and Wen-Song Tan contributed to the conception, design of the work or of parts of it, and its interpretation.


This research was supported by the Basic Research Project of Shanghai Science and Technology Commission (Grant No. 16JC1400203), the National Key Research and Development Program of China, 2018YFC1105801, and the National Natural Science Foundation of China (Grant No. 81671841).

Compliance with Ethical Standards

Conflict of interest

The authors have declared that no conflict of interest exists.


  1. 1.
    Martin P, Friedman LS. Assessment of liver function and diagnostic studies. In: Handbook of Liver Disease. 2018:1–17.Google Scholar
  2. 2.
    Hernaez R, Solà E, Moreau R, et al. Acute-on-chronic liver failure: an update. Gut. 2017;66:541–553.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Habib S, Shaikh OS. Drug-induced acute liver failure. Clin Liver Dis. 2017;21(1):151–162.PubMedCrossRefGoogle Scholar
  4. 4.
    Bernal W, Wendon J. Acute liver failure. N Engl J Med. 2013;369:2525–2534.PubMedCrossRefGoogle Scholar
  5. 5.
    Struecker B, Raschzok N, Sauer IM. Liver support strategies: cutting-edge technologies. Nat Rev Gastroenterol Hepatol. 2013;11:166–176.PubMedCrossRefGoogle Scholar
  6. 6.
    Sussman NL, Kelly JH. Artificial liver. Clin Gastroenterol Hepatol. 2014;12:1439–1442.PubMedCrossRefGoogle Scholar
  7. 7.
    Lee KCL, Stadlbauer V, Jalan R. Extracorporeal liver support devices for listed patients. Liver Transpl. 2016;22(6):839–48.PubMedCrossRefGoogle Scholar
  8. 8.
    Yu CB, Pan XP, Li LJ. Progress in bioreactors of bioartificial livers. Hepatobiliary Pancreat Dis Int. 2009;8:134–140.PubMedGoogle Scholar
  9. 9.
    Gu J, Shi X, Ren H, et al. Systematic review: extracorporeal bio-artificial liver-support system for liver failure. Hep Intl. 2012;6:670–683.CrossRefGoogle Scholar
  10. 10.
    Pan XP, Li LJ. Advances in cell sources of hepatocytes for bioartificial live. Hepatobiliary Pancreat Dis Int. 2012;11:594–605.PubMedCrossRefGoogle Scholar
  11. 11.
    Huang P, He Z, Ji S, et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature. 2011;475:386–389.PubMedCrossRefGoogle Scholar
  12. 12.
    Huang P, Zhang L, Gao Y, et al. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell. 2014;14:370–384.PubMedCrossRefGoogle Scholar
  13. 13.
    Shi XL, Gao Y, Yan Y, et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Res. 2016;26:206.PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Valk JVD, Brunner D, Smet KD, et al. Optimization of chemically defined cell culture media—replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro. 2010;24:1053–1063.PubMedCrossRefGoogle Scholar
  15. 15.
    Gu C, Li PP, Liu W, et al. The role of insulin in transdifferentiated hepatocyte proliferation and function in serum-free medium. J Cell Mol Med. 2019;23:4165–4178.PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Blüml Gerald. Microcarrier cell culture technology. Methods Biotechnol. 2007;24:149–178.CrossRefGoogle Scholar
  17. 17.
    by Vero Cells Grown on Cytodex 1 Microcarriers in a 2-Litre Stirred Tank Bioreactor. J Biomed Biotechnol. 2015;2010:586363.Google Scholar
  18. 18.
    Fernandes AM, Fernandes TG, Diogo MM, et al. Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system. J Biotechnol. 2007;132:227–236.PubMedCrossRefGoogle Scholar
  19. 19.
    Tao X, Shaolin L, Yaoting Y. Preparation and culture of hepatocyte on gelatin microcarriers. J Biomed Mater Res, Part A. 2010;65:306–310.Google Scholar
  20. 20.
    Schulz CM, Ruzicka J. Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor. The Analyst. 2002;127:1293–1298.PubMedCrossRefGoogle Scholar
  21. 21.
    Werner A, Duvar S, Müthing Johannes, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. Biotechnol Bioeng. 2000;68:59–70.PubMedCrossRefGoogle Scholar
  22. 22.
    Gstraunthaler G, Lindl T, et al. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65:791–793.PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Li PP, Gu C, Liang BY, et al. A serum-free medium suitable for maintaining cell morphology and liver-specific function in induced human hepatocytes. Cytotechnology. 2019;71:329–344.PubMedCrossRefGoogle Scholar
  24. 24.
    Dohi N, Takahashi T, Minekawa K, et al. Power consumption and solid suspension performance of large-scale impellers in gas–liquid–solid three-phase stirred tank reactors. Chem Eng J. 2004;97:103–114.CrossRefGoogle Scholar
  25. 25.
    Frijlink JJ, Bakker A, Smith JM. Suspension of solid particles with gassed impellers. Chem Eng Sci. 1990;45:1703–1718.CrossRefGoogle Scholar
  26. 26.
    Croughan MS, Hamel JF, Wang DIC. Hydrodynamic effects on animal cells grown in microcarrier cultures. Biotechnol Bioeng. 2000;67:841–852.PubMedCrossRefGoogle Scholar
  27. 27.
    Jiang D, Hu J, Zhou Y, et al. Optimization of attachment conditions for rabbit mesenchymal stem cells in cytodex 3 microcarrier culture systems. Shengwu yixue gongchengxue zazhi. 2007;24:884.PubMedGoogle Scholar
  28. 28.
    Shiojiri N, Sugiyama Y. Immunolocalization of extracellular matrix components and integrins during mouse liver development. Hepatology. 2004;40:346–355.PubMedCrossRefGoogle Scholar
  29. 29.
    Nienow AW, Hewitt CJ, Heathman TRJ, et al. Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms. Biochem Eng J. 2016;108:24–29.CrossRefGoogle Scholar
  30. 30.
    Burnouf T, Griffiths E, Padilla A, et al. Assessment of the viral safety of antivenoms fractionated from equine plasma. Biologicals. 2004;32:115–128.PubMedCrossRefGoogle Scholar
  31. 31.
    Yi G, Huanzhang H, Ke C, et al. Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates. World J Gastroenterol. 2000;6:365–370.CrossRefGoogle Scholar
  32. 32.
    Yiheng C, Shuyu T, Xuping L, et al. The effects of microcarrier concentration and cell density on the growth of swine testicle cells. Biotechnol Bull. 2016;32:242–250.Google Scholar
  33. 33.
    Demetriou AA, Reisner A, Sanchez J, et al. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology (Baltimore, Md.),. 1988;8:1006–1009.CrossRefGoogle Scholar
  34. 34.
    Hewitt CJ, Lee K, Nienow AW, et al. Expansion of human mesenchymal stem cells on microcarriers[J]. Biotech Lett. 2011;33:2325–2335.CrossRefGoogle Scholar
  35. 35.
    Shin WY, Lee KU, Lee HW, et al. Optimal number of hepatocytes per microcarrier in spheroid culture using cytodex 3 microcarrier. J Korean Surg Soc. 2007;73:235–241.Google Scholar
  36. 36.
    Liu ML, Mars WM, Zarnegar R, et al. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology. 2010;19:1521–1527.CrossRefGoogle Scholar
  37. 37.
    Grinnell F, Hays DG, Minter D. Cell adhesion and spreading factor: partial purification and properties. Exp Cell Res. 1977;110:175–190.PubMedCrossRefGoogle Scholar
  38. 38.
    Hughes RC, Pena SDJ, Clark J, et al. Molecular requirements for the adhesion and spreading of hamster fibroblasts. Exp Cell Res. 1979;121:307–314.PubMedCrossRefGoogle Scholar
  39. 39.
    Feinberg AW, Schumacher JF, Brennan AB. Engineering high-density endothelial cell monolayers on soft substrates. Acta Biomater. 2009;5:2013–2024.PubMedCrossRefGoogle Scholar
  40. 40.
    Matsuo M, Sakurai H, Ueno Y, et al. Activation of MEK/ERK and PI3K/Akt pathways by fibronectin requires integrin αv-mediated ADAM activity in hepatocellular carcinoma: a novel functional target for gefitinib. Cancer Sci. 2006;97:155–162.PubMedCrossRefGoogle Scholar
  41. 41.
    Illario M, Cavallo AL, Monaco S, et al. Fibronectin-induced proliferation in thyroid cells is mediated by αvβ3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals. J Clin Endocrinol Metab. 2005;90:2865–2873.PubMedCrossRefGoogle Scholar
  42. 42.
    Gigout A, Buschmann MD, Jolicoeur M. Chondrocytes cultured in stirred suspension with serum-free medium containing pluronic-68 aggregate and proliferate while maintaining their differentiated phenotype. Tissue Eng Part A. 2009;15:2237–2248.PubMedCrossRefGoogle Scholar
  43. 43.
    Huang L, Xiao L, Jung Poudel A, et al. Porous chitosan microspheres as microcarriers for 3D cell culture. Carbohyd Polym. 2018;202:611–620.CrossRefGoogle Scholar
  44. 44.
    Rebelo SP, Costa R, Silva MM, et al. Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures. J Tissue Eng Regen Med. 2017;11:2034–2045.PubMedCrossRefGoogle Scholar
  45. 45.
    Wei G, Wang J, Lv Q, et al. Three-dimensional coculture of primary hepatocytes and stellate cells in silk scaffold improves hepatic morphology and functionality in vitro. J Biomed Mater Res, Part A. 2018;106:2171.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiPeople’s Republic of China

Personalised recommendations