文献类型：期刊文献

英文题名：Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System

作者：Gu, Ce[1];Chai, Miaomiao[1];Liu, Jiaxing[1];Wang, Hui[1];Du, Wenjing[1];Zhou, Yan[1];Tan, Wen-Song[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2020

卷号：65

期号：7

起止页码：2009

外文期刊名：DIGESTIVE DISEASES AND SCIENCES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000496229700004)】；

基金：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).

语种：英文

外文关键词：Transdifferentiated hiHeps; BAL; Serum-free medium; Microcarrier culture; Fibronectin

摘要：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. Methods 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. Results 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 x 10(5) 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 x 10(6) 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-beta 1/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. Conclusions Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.