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Updated: Jun 3, 2026
Isolation and Primary Culture of Rat Hepatic Cells
Published on: June 29, 2012
[In vitro proliferation of rat Leydig cells]
Hong-da Bi1, Xiao-yun Wang, Guang-dong Zhou
1Department of Plastic Surgery, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China. bihongda0411@yahoo.com.cn
Objective:
To investigate the feasibility of in vitro proliferation of rat Leydig cells by modifying the cell culture system.
Methods:
Leydig cells were isolated from three-week-old rats by a procedure combining collagenase dispersion, stainless steel mesh infiltration and differential adhesion. The isolated cells were cultured in DMEM/F12 and modified media for stem cell proliferation, and the proliferation of the cultured cells was evaluated by cell counting and MTP test. The expression of 3beta-HSD in the cultured cells was detected by immunohistochemistry and flow cytometry, and testosterone productivity in the isolated Leydig cells with or without hCG stimulation was determined at 2 hours and 4 days after cell isolation.
Results:
The Leydig cells cultured in the modified media proliferated actively, with a doubling time of (2.26 +/- .31) days, as compared with (16.32 +/- 2.14) days for those cultured in the traditional media (P <0.05). The 3beta-HSD positive rate in the cultured cells was (554.3 +/- 7.1)% after 2 hours and (93.6 +/- 4.6)% after 4 days of culture. All the proliferated cells exhibited testosterone productivity, and their testosterone secretion was significantly upregulated by hCG stimulation (P <0.05).
Conclusion:
Leydig cells isolated by differential adhesion proliferate actively in the modified culture media.
