Development of <i>in vitro</i> 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 × 10 6 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 × 10 6 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.

Perkembangan Sel Endometrium Domba setelah Inkubasi dalam Kolagenase dan Dikultur In Vitro dengan Estradiol dan Progesteron

“…Beberapa peneliti lainnya juga menggunakan enzim kolagenase untuk mengisolasi sejumlah sel seperti sel kondrosit (Yonenaga et al, 2017), sel mesenkim pada tali pusar anjing (Lee et al, 2013), sel Leydig pada tikus (Kaiin et al, 2013) sel germinal pada testis kuda (Jung dan Yoon 2016), sel Schwann (Jin et al, 2008) serta progenitor sel otot (Ishii et al, 2017). Sementara itu, penelitian tentang lama waktu inkubasi enzim kolagenase untuk mendapatkan sejumlah sel endometrium bervariasi baik tentang lama inkubasi maupun hasil yang dilaporkan seperti pada kelinci dengan lama waktu inkubasi 1,5 jam (Mulholland et al, 1988), sapi dengan lama waktu inkubasi dua jam (Kimmins et al, 2003), mencit dengan lama waktu inkubasi 20-30 menit (Wewer et al, 1986) hingga 1,5 jam (Janzen et al, 2013), anjing dengan lama waktu inkubasi 4-6 jam (Bartel et al, 2013), babi dengan lama waktu inkubasi yang berbeda yaitu satu jam (Wang et al, 2000), 1,5 jam (Blitek dan Ziecik, 2004), dan 2 jam (Aldarmahi, 2017).…”