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- Title
Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse.
- Authors
Thanh Minh Dang; Trinh Van Le; Huy Quang Do; Van Thuan Nguyen; Ai Xuan Le Holterman; Loan Tung Thi Dang; Nhan Chinh Lu Phan; Phuc Van Pham; Son Nghia Hoang; Long Thanh Le; Gabriele Grassi; Nhung Hai Truong
- Abstract
Liver fibrosis (LF) mortality rate is approximately 2million 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.
- Subjects
LIVER cells; OLEIC acid; PHENOTYPES; MICE; FLOW cytometry; GLUTAMINE synthetase; ACYLTRANSFERASES
- Publication
Bioscience Reports, 2021, Vol 41, Issue 1, p1
- ISSN
0144-8463
- Publication type
Article
- DOI
10.1042/BSR20202514