Development of Efficient Cryopreservation Method for Spermatogonial Stem Cells Using Trehalose.

Abstract

Spermatogenesis is a complex and very efficient process that supported by spermatogonial stem cells (SSCs). Since SSCs are the only cells in the adult mammalials body that undergoes division and can transmit male genetic information to subsequent generation, the study about their cryopreservation would provide the valuable resource for industries related to human medical research, animal husbandry, conservation and genetic modification. The aim of this study was to find an efficient cryopreservation protocol for SSCs.Testis cells for SSCs culture were collected from a pup transgenic mouse line C57BL/6-TgEGFP that was bred into DBA/2 background (designated Green) and SSCs from the collected cells purified via Magnetic Activated Cell Sorting (MACS) technique with anti-Thy-1 antibody after enzymatic digestion. After 4 wk of initiation of culture, highly purified SSCs were collected, and then, to determine the effect of trehalose on the cryopreservation of SSCs, we added the trehalose at 50, 100 and 200mM in the basal freezing medium (MEM alpha containing 10% dimethly sulphoxide and 10% fetal bovine serum) and the frozen cells were preserved in liquid nitrogen. 1 wk, 1 mo., 3 mo. after freezing, survival rate and normalize rate of proliferation were compared among the concentrations of trehalose. When the survival rate was determined, only significant improvement was obtained in the 50mM trehalose treated group (1 wk, 1 mo. and 3 mo. are 89.7 ± 2.0, 85.2 ± 1.8, 86.1 ± 1.2% respectively) at all freezing-period compared with controls (1 wk, 1 mo. and 3 mo. are 76.1 ± 3.4, 69.1 ± 2.3, 68.8 ± 1.5%; p < 0.05). Thus, 50mM trehalose treated group (79.0 ± 1.8%) obtained the significant improvement of proliferation rate compared with control (54.4 ± 4.4%) after freezing 1wk. However, after freezing 1 mo. and 3 mo., the significant improvement of proliferation rate was showed in 200mM trehalose treated group (1 mo. and 3 mo. are 59.3 ± 3.2, 49.1 ± 3.9% respectively) compared with control (1 mo. and 3 mo. are 42.2 ± 0.6, 27.5 ± 0.5% respectively; p < 0.05). Furthermore, after freezing 1mo. and 3 mo., apoptotic cells rate of 200mM trehalose treated group was significantly lower than other groups. Therefore, 200mM trehalose treated group was selected for subsequent experiments. To analyze the effect of storage length on the proliferation of 200mM trehalose treated group, we additionally determined the proliferation rate to 6 mo-12 mo. after freezing. Although proliferation of freeze-thawed SSCs steadily decreased to 3 mo., no decrease was shown after 3 mo. The number of colonies of 200mM trehalose treated group that was frozen during 3 mo. (506 ± 72.8) was significantly higher than trehalose non-treated group (238 ± 28.0; p < 0.05). When 200mM trehalose treated group was transplanted to infertile recipient, complete spermatogenesis derived from freeze-thawed SSCs occurred in the recipient mouse testes, and normal offspring were obtained from recipients by natural mating with wild type female mice. The results indicated that 200mM trehalose is effective cryoprotectant as additives to freezing medium for SSCs. This study was supported by a grant of the Ministry of Education, Science and Technology (2010-0023848), Ministry for Food, Agriculture, Forestry and Fisheries (110025-03-1-HD110), Rural Development Administration (No.PJ008048).