Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse.

Autor:	Dang TM; Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam.; School of Biotechnology, International University, VNUHCM, Ho Chi Minh City, Vietnam., Le TV; Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam., Do HQ; Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam., Nguyen VT; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam.; School of Biotechnology, International University, VNUHCM, Ho Chi Minh City, Vietnam., Holterman AXL; Department of Pediatrics and Surgery, University of Illinois College of Medicine, Chicago and Peoria, Illinois, U.S.A., Dang LTT; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam.; Faculty of Biology and Biotechnology, University of Science, VNUHCM, Ho Chi Minh City, Vietnam., Phan NCL; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam.; Stem Cell Institute, University of Science, VNUHCM, Ho Chi Minh City, Vietnam., Pham PV; Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam., Hoang SN; Animal Biotechnology Department, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam., Le LT; Animal Biotechnology Department, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam., Grassi G; Department of Life Sciences, Cattinara University Hospital, Trieste University, Trieste, Italy., Truong NH; Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.; University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam.; Faculty of Biology and Biotechnology, University of Science, VNUHCM, Ho Chi Minh City, Vietnam.
Jazyk:	angličtina
Zdroj:	Bioscience reports [Biosci Rep] 2021 Jan 29; Vol. 41 (1).
DOI:	10.1042/BSR20202514
Abstrakt:	Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC's undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture. (© 2021 The Author(s).)
Databáze:	MEDLINE
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