Study of In Vitro Cell Culture and The Role of Rat Leydig Cells Conditioned Medium on Bone Marrow Mesenchymal Stem Cells Differentiation. Supervised

Abstract

The use of Percoll gradients for Leydig cells isolation has been reported to influence cell toxicity and reduce the viability and the amount of living cells after culture. To dissolve this problem, study has been carried out used non toxic Nycodenz gradient to isolated and purified the Leydig cells. The use of Nycodenz gradient for the isolation and purification of Leydig cells has not been done by other researchers. To obtain the optimum condition of Leydig cells production, growth factor is required as supporting factors for development of growing cells. Therefore, hCG and ITS has been used in this study. Leydig cells conditioned medium (LCM) contained bioactive materials such as testosterone and growth factors. The Leydig cells conditioned medium as culture medium for bone marrow mesenchymal stem cells will be used to determine its influence to differentiation of cells. The phase of this study included: 1. Isolation and purification of Leydig cells using Nycodenz gradient : (I) with 3 column ( 5 % , 10 % , 15 % ) and ( II ) with 5 columns ( 4 % , 8 % , 10 % , 12 % , 15 % ) and Percoll gradient ( 21 % , 26 % , 34 % , 40 % , 60 % ) as a control . 2. In vitro culture of the best result from cell purification was cultured with the addition of hormones and growth factors : (1) DMEM+NBCS 10% as control, (2) DMEM + NBCS + 2.5 IU/ml hCG , (3) DMEM + NBCS+ 5 ug / ml insulin, 10 ug/ml transferrin, selenium 5 ug/ml (ITS ) and (4) DMEM +NBCS + hCG+ITS . 3 . The concentration of testosteron and protein analyzed from Leydig cells conditioned medium has been used for culturing bone marrow mesenchymal stem cells. The result of first phase study was Nycodenz gradient have the same purity and viability compared with Percoll gradient. After 3 days cultured, in Nycodenz gradient the concentration and viability of Leydig cells more higher than Percoll gradient. The combination of hCG and ITS in DMEM produced Leydig cell proliferation (90.64%) higher compared with hCG (86.99%), ITS (88.35%) and control (86.82%). Proliferation rate (population doubling time, PDT) from primary culture using combination of hCG and ITS showed the fastest (0.88 day) compared with ITS (0.97 day), hCG (1.02 day) and control (1.03 day). The same result of Leydig cells proliferation rate has also showed in first and second cell lines. Testosterone consentration analyzed was the highest (5.25 ng/ml) in DMEM combination with hCG and ITS compared with other treatments (hCG 5.06 ng/ml; ITS 3.19 ng/ml and control 2.46 ng/ml). Differentiation to became Leydig cells from bone marrow mesenchymal stem cells has been known from positive reaction to 3β-HSD staining. Added of hCG to Leydig cells conditioned medium was able to increased the number of Leydig cells to 71.9%. It was also showed that concentration of testosterone has been increased (0.93 ng/ml and 10.22 ng/ml) in the culture medium. The combination of hCG and ITS in DMEM had testosterone (0.71 ng/ml) and protein concentration (985.29 μg/ml) higher than the other treatments. The Leydig cell conditioned medium in this study had three protein bands with molecular weight (MW) approximately to 29 kDa, 62-70 kDa and 95-130 kDa. Based on these results it could be concluded that the use of Nycodenz gradient II was effective to purified of Leydig cells. The combination of hCG and ITS was the best result to increase proliferation and testosterone. Leydig cells conditioned medium could also to differentiate mesenchymal stem cells into Leydig cells (transdiferentiation).