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

Author:

Dang Thanh Minh123,Le Trinh Van12,Do Huy Quang12,Nguyen Van Thuan23,Holterman Ai Xuan Le4,Dang Loan Tung Thi25,Phan Nhan Chinh Lu26,Pham Phuc Van12,Hoang Son Nghia7,Le Long Thanh7,Grassi Gabriele8,Truong Nhung Hai125ORCID

Affiliation:

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

Abstract

Abstract 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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry,Biophysics

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3