fa Morphometry Morphometry Original Original <strong>Purpose:</strong> To investigate of the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on proliferation of frozen-thawed neonate mouse spermatogonial stem cells.<strong>Materials and Methods</strong>: 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, GFR&alpha;-1, VASA, ITGA6 and ITGB1). The significancy of the data was analyzed using Repeated Measures and ANOVA tests.<strong>Results</strong>: 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&plusmn;2.2 and 63&plusmn;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&pound;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&pound;0.01). There was a significant down-regulation of spermatogonial genes in the frozenthawed groups after three weeks of culture.<strong>Conclusions</strong>: The spermatogonial cells seeding on PLLA can increase in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells. Spermatogonil Stem Cells, Plla Nano Fibers, Testis 0 0 http://anatomyjournal.ir/browse.php?a_code=A-10-304-239&slc_lang=fa&sid=1 Neda Slahi 10031947532846006327 10031947532846006327 Yes Mahmoud Reza Hadjighassem 10031947532846006328 10031947532846006328 No Mohammad Taghi Joghataei 10031947532846006329 10031947532846006329 No Mehrdad Bakhtiyari 10031947532846006330 10031947532846006330 No Mahmoud Ayyoubiyan 10031947532846006331 10031947532846006331 No Mohammad Hossein Asadi 10031947532846006332 10031947532846006332 No Seyyed Morteza Koruji 10031947532846006333 10031947532846006333 No