P–798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis
Authors: Tang S , Jones C , Coward K 
Published: August 2021

Abstract

Study question
Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis?

Summary answer
Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study.

What is known already
Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed.

Study design, size, duration
This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements.

Participants/materials, setting, methods
Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP–9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα–1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR).

Main results and the role of chance
The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (&lt;10% SSCs) and enriched SSCs(&gt;50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture.

Limitations, reasons for caution
We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells.
Wider implications of the findings: Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future.

Trial registration number
University of Oxford