Evaluation of alginate hydrogel cytotoxicity on three-dimensional culture of type A spermatogonial stem cells.

The culture of spermatogonial cells for future transplantation, based on the specific biology of these cells is important and necessary. Recently, the use of scaffolds especially alginate for culturing stem cells has been the focus of many researchers. The aim of this study was to evaluate the cytotoxicity of alginate hydrogels to cultures of type A spermatogonial stem cells. Spermatogonial stem cells of 6day-old immature mice were isolated by surgery; thereafter, the cells were purified by MACS using antibodies against thy-1 and C-kit and cultured on a layer of laminin. After purification, spermatogonial stem cells were encapsulated in alginate hydrogels. After one month of encapsulation and culture in DMEM culture medium containing 10ng/ml GDNF, cells were removed from hydrogel and were examined for viability, cell morphology and structure, cytotoxicity and expression of apoptosis genes Fas, P53, Bax, Bcl2, Caspase3 by staining with trypan blue, scanning electron microscopy, LDH test, and Real time PCR, respectively. The encapsulation did not change the morphology and viability of spermatogonial stem cells. Investigations showed that spermatogonial stem cells preserve by the high viability (74.08%) and cytotoxicity of alginate hydrogel was estimated to be 5%. Expression of Fas gene increased in main group compared with the control group, and expression of Bax and P53 was reduced in main group compared with the control group. Expression of Bcl2 and Caspase3 genes did not show any significant difference between the main group and the control group. Considering the lack of cytotoxicity and antioxidant properties of alginate hydrogel scaffold and high viability of cells, this three-dimensional scaffold is applicable for culturing and encapsulation of spermatogonial stem cells.