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Background The present study was designed to evaluate the survival and proliferation of spermatogonial stem cells from cryptorchid mouse testis in co-culture system over a 3 weeks period. Materials and methods Sertoli and spermatogonial cells were isolated from bilateral cryptorchid mouse model testes. Isolated spermatogonial cells were co-cultured with Sertoli cells in minimal essential medium (α-MEM) supplemented with 10% fetal calf serum (FCS) for three weeks. The identity of the cells was confirmed through immunocytochemistry against Oct-4 and Vimentin. Results Best results were achieved from the co-culture system spermatogonia which continued to proliferate, and eventually, type A spermatogonia colonies were found. Most of the cells in these colonies were Oct-4 positive. Conclusion Bilateral cryptorchid surgery model is a good model for enrichment of spermatogonial stem cells (SSCs). These cells can be used for molecular characterization, genetic manipulation and restoration of male fertility.