Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System.
Dig Dis Sci
; 65(7): 2009-2023, 2020 07.
Article
em En
| MEDLINE
| ID: mdl-31722057
ABSTRACT
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 × 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-ß1/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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas de Cultura de Células
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Fígado Artificial
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Hepatócitos
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Proliferação de Células
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Transdiferenciação Celular
Limite:
Humans
Idioma:
En
Revista:
Dig Dis Sci
Ano de publicação:
2020
Tipo de documento:
Article