Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix

Abstract

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.

Organoids: a promising new in vitro platform in livestock and veterinary research

“…Two studies reported the production of porcine testicular “organoids” from dissociated testis [ 44 , 45 ]. The developed testicular structures contained all relevant cell types in a 3D organization with physiological cell–cell interactions of germ cells with supporting cells [ 44 ].…”

“…In addition, the cultured testicular structures contained Sertoli cells and germ cells which assembled into seminiferous tubule-like structures delimited by a basement membrane along with Leydig cells and peritubular cells. In one of these studies, the cellular structures were maintained for 45 days [ 45 ]. However, long term self-renewal was not well-controlled, as the number of Sertoli cells increased and that of Leydig cells decreased over time.…”

“…However, the 3D cultures in some of the earlier papers listed in Table 1 do seem to share certain features with self-renewing organoids, describing differentiation of organ-like structures from progenitor cells using ECM components and growth factors to induce 3D tissue development. Two studies reported the production of porcine testicular "organoids" from dissociated testis [44,45]. The developed testicular structures contained all relevant cell types in a 3D organization with physiological cell-cell interactions of germ cells with supporting cells [44].…”

Proteinaceous Hydrogels for Bioengineering Advanced 3D Tumor Models

“…[ 116,119 ] Nonetheless, dECM‐based hydrogels are still relatively underexplored for developing in vitro tumor models. [ 120–122 ]…”

Polymer Hydrogels to Guide Organotypic and Organoid Cultures

“…Porcine testicular organoids obtained from testicular single cells [ 130 ] embedded in tissue‐specific dECM gels underwent seminiferous tubule organization comparable to the native organ. [ 131 ] The culture of freshly isolated, pre‐aggregated testicular cells in the very soft decellularized testicular dECM gel (storage modulus below 20 Pa) was clearly superior to the reference culture in collagen hydrogels (storage modulus 75–100 Pa). The dECM gel‐grown testicular organoids were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality.…”