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 OJAS  Vol.3 No.4 , October 2013
Development of in vitro culture of rat Leydig cells after purification with Nycodenz gradient
Abstract: The study examined the effect of human Chorionic Gonadotrophin (hCG) and/or Insulin Transferring Sodium Selenite (ITS) on the proliferation and development of rat Leydig cells purified by Nycodenz gradient. Leydig cells purity, viability and proliferation after purification and 3 days of cultured were evaluated. Leydig cells 1 × 106 cells/ml were cultured in DMEM containing 10% Newborn Calf Serum (NBCS) and divided into four kinds of treatments 1) as a control, 2) control + 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin, 10 μg/ml transferrin, 5 μg/ml Se), and 4) control + hCG + ITS. Leydig cells purification results showed 91.40% of purity, viability was 98.17% and concentration 7.03 × 106 cells/ml. The addition of ITS and hCG + ITS in DMEM produced Leydig cell proliferation by 88.35% and 90.64% higher than in controls (86.82%) (p < 0.05). The addition of hCG did not increase Leydig cell proliferation (86.99%). In the primary culture of Leydig cells, the population doubling time (PDT) was 1.03 days, similar with the addition of hCG treatment (1.02 day). Declined in value of PDT significantly (p < 0.05) occurred in DMEM supplemented with ITS (0.97 day) and hCG + ITS (0.88 day). This result was also seen in the first and second Leydig cell lines. DMEM added with hCG or hCG + ITS resulted in a higher amount of testosterone (5.06 ng/ml; 5.25 ng/ml)) than the culture medium without hCG (2.46 ng/ml) (p < 0.05). It can be concluded that the combination of hCG + ITS can increase the number of Leydig cells and testosterone levels in the culture medium.
Cite this paper: Kaiin, E. , Djuwita, I. , Yusuf, T. and Setiadi, M. (2013) Development of in vitro culture of rat Leydig cells after purification with Nycodenz gradient. Open Journal of Animal Sciences, 3, 299-304. doi: 10.4236/ojas.2013.34044.
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