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

Summary Proliferation of spermatogonial stem cells ( SSC s) in vitro system is very important. It can enhance SSC s 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 SSC s were co‐cultured on fresh S ertoli cells (experimental group 1), and frozen‐thawed S ertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSC s were co‐cultured on fresh S ertoli 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 SSC s for men who have a cancer‐related illness and waiting for radiotherapy and/or chemotherapy.