Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation.

Guillaume Richer,Robin M Hobbs,Katherine L Loveland,Ellen Goossens,Yoni Baert

doi:10.3389/fphys.2021.757565

Guillaume Richer, Robin M Hobbs + Show 3 more

Open Access

https://doi.org/10.3389/fphys.2021.757565

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Abstract

Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.

Highlights

Spermatogenesis is the stepwise process of sperm development within the seminiferous tubules of the testis
Tubule-like structures of testicular organoids (TOs) in 1LSs from both strains displayed similar somatic marker spatial arrangements, resembling their in vivo expression profiles (Supplementary Figures 1B–E): Leydig (3ß-HSD+) and elongated peritubular myoid (ACTA2+) cells reorganized around the basement membrane (LN+) of tubules containing Sertoli cells (SOX9+) that were interconnected by tight junctions (ZO1+; Figures 2E–H)
3D printed macropores served as a delimitation of the area in which testicular cells can grow in order to optimize TO morphology and histology

Summary

Introduction

Spermatogenesis is the stepwise process of sperm development within the seminiferous tubules of the testis. The tubules are delimited by a basement membrane and contractile myoid cells. Sertoli cells directly nurture germ cells through the different stages of spermatogenesis: from spermatogonial stem cell (SSC) at the basement membrane to post-meiotic spermatids toward the lumen. The fate of germ cells relies on secretions coming from the interstitial compartment between the tubules such as testosterone produced by the Leydig cells (Oliver and Stukenborg, 2019). Because of the highly coordinated nature of signaling cues in the testicular microenvironment, it is pivotal for cultures for in vitro spermatogenesis (IVS) to reestablish conditions that mimic the two structural compartments of the testis (Oliver and Stukenborg, 2019).

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