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Evaluation of the effects of cryopreservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n = 8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen‐thawed SSCs were co‐cultured on fresh Sertoli cells (experimental group 1), and frozen‐thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co‐cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen‐thawed testicular cells after 2 weeks of culture had a significantly (P < 0.05) higher percentage of living cells compared to frozen‐thawed testicular cells at the beginning of culture (59.2 ± 7.05 and 46.3 ± 8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6 ± 2.8 and 8.33 ± 1.5, respectively, P < 0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P < 0.05) after 3 weeks of culture (269.7 ± 52.1, 204.34 ± 24.1 and 112.52 ± 23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer‐related illness and waiting for radiotherapy and/or chemotherapy.