The Effects of Plla Nanofiber Scaffold on Proliferation of Frozen-Thawed Neonate Mouse Spermatogonial Stem Cells

Purpose: To investigate of the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on proliferation of frozen-thawed neonate mouse spermatogonial stem cells.Materials and Methods: Spermatogonial cells were isolated from neonatal 3-6-day-old NMRI mice testes by two steps enzymatic digestion and differential plating. The isolated spermatogonial cells were divided into four culture groups: 1) fresh spermatogonial cells, 2) fresh spermatogonial cells seeded onto PLLA 3) frozen-thawed spermatogonial cells, 4) frozen-thawed spermatogonial cells seeded onto PLLA. Cells in all groups were cultured in DMEM supplemented with 5% FCS and 10 ng/ml GDNF for 3 weeks. Diameter and number of clusters which were determined during the culture and semi-quantitative RT-PCR were carried out at the end of 3rd week for all culture groups. Presence of spermatogonia at the culture was determined by reverse transcription polymerase chain reaction (RT-PCR) for several important spermatogonial markers (PLZF, Oct4, GFRα-1, VASA, ITGA6 and ITGB1). The significancy of the data was analyzed using Repeated Measures and ANOVA tests.Results: The findings indicated that the viability rate of the fresh cell (control 1 and exprimental 1) and the frozen cells after thawing (control 2 and exprimental 2) were 89.25±2.2 and 63±3.56, respectively and the differences were significant (p<0.001). In vitro culturing of spermatogonial cells on PLLA significantly increased the formation of cell clusters in comparison with those of the control groups (p£0.001). Although the differences of the diameters of clusters in the fresh cell groups were not significant, culturing of frozen-thawed cells on PLLA significantly decreased their diameters (p£0.01). There was a significant down-regulation of spermatogonial genes in the frozenthawed groups after three weeks of culture.Conclusions: The spermatogonial cells seeding on PLLA can increase in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells.