Establishment and characterization of a porcine testis organoid culture system

Abstract

The studies presented in this thesis were designed to establish, examine and characterize a porcine testis organoid culture system. The objective of the first study was to establish a porcine testis organoid system and assess different culture conditions for its efficient in vitro morphogenesis. We first examined the potential of testis cells from 1-week-old piglets for de novo testis organogenesis (i.e., formation of testis organoids) using spheroid-forming low attachment wells in an air-liquid interface culture system. We found that both fresh and cryopreserved neonatal porcine testis cells were able to self-reassemble into testis organoids comprising of testis cord-like structures which could be maintained for at least 4 weeks in culture. Next, we examined several culture conditions including different cell densities, culture durations, media supplementation, and germ cell (gonocyte) ratios to find the optimal conditions for de novo tubulogenesis in testis organoids. We found that a testis cell density of 0.8×106 cells/organoid and a combined supplementation of 5% knockout serum replacement (KSR) and 10% fetal bovine serum (FBS) in media would improve de novo formation of testis cord-like structures. We also observed that the relative number of germ cells in organoids did not decrease over time or differ among supplementation groups. The objective of the second study was to examine the structural and endocrine characteristics of our newly developed porcine testis organoids. We found that testis organoids consisted of tubular and interstitial compartments resembling innate testis tissue. More specifically, germ cells were observed within the testis tubular compartment reconstructed by Sertoli cells, peritubular myoid cells (PTMCs), and a peritubular basement membrane. We also observed the reconstruction of the inter-tubular interstitial compartment containing Leydig cells, collagen fibers, and vascular structures. Furthermore, we showed that testis organoids respond to luteinizing hormone (LH) stimulation and secrete testosterone, suggesting that the organoids also possess the endocrine characteristics of a typical neonatal testis tissue. This study provides a platform for the production of testis organoids, with high structural and functional resemblance to the innate neonatal testis tissue. The new testis organoid system can be used in future studies into de novo testis organogenesis or in pharmaco-toxicological testing of various substances on testis formation.