Testicular organoid generation by a novel in vitro three-layer gradient system

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche in vitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the in vivo germ-to-somatic cell associations in vitro. Moreover, we also verified the roles of retinoic acid (RA), IL-1 alpha, TNF alpha and RA inhibitors in germ cell maintenance and BTB organization in vitro. Treatment with RA was beneficial for germ cell maintenance, while IL-1 alpha and TNF alpha were observed to impair the formation of testicular organoids and germ cell maintenance. Taking in account our characterization and validation results, we propose the 3-LGS as a new platform to investigate the SSC niche in vitro and to search for novel unknown factors involved in germ cell proliferation and differentiation. Moreover, we suggest that this model can be used in other scientific fields to study organogenesis and development by the generation of organoids. (C) 2017 The Author(s). Published by Elsevier Ltd.