[ effects of PLLA nanofiber scaffold on proliferation of frozen-thawed neonate mouse spermatogonial stem cells]

ABSTRACT

To investigate of the effects of a poly L-lactic acid [PLLA] nanofiber scaffold on proliferation of frozen-thawed neonate mouse spermatogonial stem cells. 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, GFRalpha-1, VASA, ITGA6 and ITGB1]. The significancy of the data was analyzed using Repeated Measures and ANOVA tests. 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 [pcell groups were not significant, culturing of frozen-thawed cells on PLLA significantly decreased their diameters [pdown-regulation of spermatogonial genes in the frozenthawed groups after three weeks of culture. The spermatogonial cells seeding on PLLA can increase in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells