Colonization of Mouse Spermatogonial Cells in Modified Soft Agar Culture System Utilizing Nanofibrous Scaffold: A New Approach

Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study
aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a
new approach to mimic in vivo conditions of SSCs development.

The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and
covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed
in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and
c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining.

Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P<0.05). Only Plzf indicated a significant
increased at the levels (P<0.05), the gene expression levels of Id4, Plzf, and Gfrα1 were higher
in the present culture system. In addition, the expression of the c-Kit gene as a differentiating
spermatogonia marker was higher in presence of scaffold and soft agar compared with the
amount of other experimental groups (P<0.05).

The culture system by using nanofibrous scaffold and soft agar as a new culture method suggests the potential of this approach in
SSCs enrichment and differentiation strategies for male infertility treatments, as well as in vitro
spermatogenesis.