effect of FBS concentration on cryopreservation of isolated spermatogonial cells from neonatal mouse by MACS method

ABSTRACT

The purpose of this study was the separation of spermatogonial cells from prepubertal NMRI mouse testis by magnetic activating cell sorting [MACS] method and assessment of FBS concentration on surviving these cells after cryopreservation. Testes from 6-days-old mouse removed and digested in two enzymatic mediums, first 8-9 min at medium containing collagenase, trypsin and DNase and second 10-12 min in medium containing collagenase, trypsin, DNase, hyalloronidase and EDTA and then the spermatogonial cells were isolated by [MACS] method. In the freezing step, the cells were frozen in three medium containing DMEM/F12 medium, 10% DMSO and 50%, 60% and 70% FBS serum, named group I, II.III. The viable cells obtained after enzymatic disaggregation were 91.66% +/- 0.60% and after being isolated by MACS method were 95.25% +/- 0.33%. The purity of the isolated cells was 93.79% +/- 2.20%. The cells frozen in group I, II and III had 39.09% +/- 0.15, 85.55% +/- 6.98 and 90/29 +/- 1/38% viability, respectively. according to the obtained results, the increase of temperature at digestion step reduces the time of testicular tissue disaggregation and consequently increase viable cells. Higher viable cells and purity can be attained by using of a6 integrin and magnetic beads. The results show that spermatogonial cells in freezing media containing 60% - 70% FBS have the highest viability after thawing