In Vitro Proliferation of Human Spermatogonial Stem Cells in Two-dimensional and Three-dimensional Culture Systems of PRP

Background: The microenvironment of SSCs has critical roles in self-renewing and differentiation ability. Platelet growth factors stimulated cell proliferation, cryoprotection, chemotaxis, and differentiation. Activation of PRP by CaC[l.sup.2] solidified plasma and forming fibrin network. Fibronectin and fibrin in PRP act as adhesion molecules that can promote cell migration and the fibrin network structure within PRP can hold cells in a 3-D organization. Here, in this study, we evaluated the effect of PRP on the self-renewing of adult human SSCs in two-dimensional and three-dimensional culture systems. Methods: Testicular cells of four brain-dead patients were cultivated, characterization of SSCs was performed and their functionality was assessed by xenotransplantation to azoospermia mice. After the preparation of the PRP scaffold, cytotoxic and histological evaluation was performed. Then, the SSCs were cultivated into three groups: control, 2-D culture by an optimized dose of PRP, and PRP scaffold. The expression of GFRa1 and c-KIT were evaluated by performing real-time PCR. Results: The expression of PLZF and GFRa1 was identified through immunocytochemistry. Labeled cells were transplanted on the seminiferous tubules basement membrane in azoospermic recipient testes as an individual cells and didn't create a cluster. This proved the existence of SSCs after 2-D pre-culture and evaluate the homing and colonization of SSCs in the recipient testis. The proliferation rate and viability of SSCs on PRP scaffolds showed a striking time-dependent increase. Histological evaluations revealed cross-linked fibrin in pink, these randomly oriented fibril organizations appeared to present an efficient microstructure for human SSCs. Expression of c-KIT showed a significant increase (P