Platelet-rich plasma hydrogel supports long-term germ cell maintenance in rat testicular organotypic culture

Fertility preservation has become an important clinical concern for prepubertal boys undergoing gonadotoxic cancer treatments. Preservation of germ cells within immature testicular tissue is therefore considered a promising strategy for future fertility restoration. The purpose of this study was to evaluate the effects of platelet-rich plasma (PRP), used as a culture medium supplement and/or as a hydrogel scaffold, on germ cell survival and spermatogenic progression in a three-dimensional rat testicular organotypic culture system and to explore its potential relevance for fertility preservation approaches.

Testicular tissue fragments obtained from postnatal day 7-9 Wistar albino rats were cultured using an air-liquid interface organotypic culture system on agarose blocks. PRP was incorporated either as a hydrogel scaffold supporting the tissue fragments or as a soluble supplement in the culture medium. Cultures were maintained for 8 weeks, and samples were collected at weeks 1, 3, 4, 6, and 8. Spermatogenic progression was evaluated using histomorphometric and immunohistochemical analyses.

SCP3( +) spermatocytes were better preserved in PRP hydrogel-supported cultures, with persistence observed up to week 8 only in this group. In addition, PLZF( +) undifferentiated spermatogonia remained detectable at week 8 in PRP hydrogel cultures, indicating improved preservation of the spermatogonial population. In vitro spermatogenesis progressed up to the spermatocyte stage in all experimental conditions, whereas differentiation into round spermatids was not observed.

This organotypic culture approach may support germ cell maintenance during long-term culture and may contribute to the development of future fertility preservation strategies.