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

  • Dang, Thanh Minh1, 2, 3
  • Le, Trinh Van1, 2
  • Do, Huy Quang1, 2
  • Nguyen, Van Thuan2, 3
  • Holterman, Ai Xuan Le4
  • Dang, Loan Tung Thi2, 5
  • Phan, Nhan Chinh Lu2, 6
  • Pham, Phuc Van1, 2
  • Hoang, Son Nghia7
  • Le, Long Thanh7
  • Grassi, Gabriele8
  • Truong, Nhung Hai1, 2, 5
  • 1 Laboratory of Stem Cell Research and Application, University of Science, VNUHCM, Ho Chi Minh City, Vietnam
  • 2 University of Science, Viet Nam National University, Ho Chi Minh City, Vietnam
  • 3 School of Biotechnology, International University, VNUHCM, Ho Chi Minh City, Vietnam
  • 4 Department of Pediatrics and Surgery, University of Illinois College of Medicine, Chicago and Peoria, Illinois, U.S.A
  • 5 Faculty of Biology and Biotechnology, University of Science, VNUHCM, Ho Chi Minh City, Vietnam
  • 6 Stem Cell Institute, University of Science, VNUHCM, Ho Chi Minh City, Vietnam
  • 7 Animal Biotechnology Department, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam
  • 8 Department of Life Sciences, Cattinara University Hospital, Trieste University, Trieste, Italy
Published Article
Bioscience Reports
Portland Press
Publication Date
Jan 22, 2021
DOI: 10.1042/BSR20202514
PMID: 33350435
PMCID: PMC7823195
PubMed Central
  • Research Articles


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.

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