Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System
- 98 Downloads
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.
KeywordsTransdifferentiated 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.Martin P, Friedman LS. Assessment of liver function and diagnostic studies. In: Handbook of Liver Disease. 2018:1–17.Google Scholar
- 17.by Vero Cells Grown on Cytodex 1 Microcarriers in a 2-Litre Stirred Tank Bioreactor. J Biomed Biotechnol. 2015;2010:586363.Google Scholar
- 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
- 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
- 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