# Human Testicular Tissue Digestion, Testicular Cell Selection, and Downstream Characterization for Reproductive Purposes: A Scoping Review

## TL;DR

This review examines methods for processing human testicular tissue to preserve fertility in childhood cancer survivors.

## Contribution

The paper systematically reviews digestion and cell selection techniques for testicular tissue, highlighting gaps and future research directions.

## Key findings

- Many digestion protocols exist, but tissue characteristics are often missing, limiting protocol comparison.
- Flow cytometry shows promise for high-purity cell selection.
- Future research should focus on digestion outcomes and cell interactions for successful in vitro spermatogenesis.

## Abstract

Fertility preservation and restoration using cryo-banked prepubertal testicular tissue is a pivotal part of the childhood hematological cancer care pathway. Estimations indicate that one in 900–1400 young adults is a childhood cancer survivor, underlying the urge to develop fertility restoration protocols as some of the patients have reached the age to father their own genetic child. While it has been reported that 39% of patients present cancer cells in their testes, no efficient decontamination technique has been identified to circumvent cancer reintroduction after autologous testicular cell transplantation. Obtaining single-cell suspensions and selecting only testicular cells might be an option. In this review, mechanical dissociation/enzymatic digestion protocols applied to human testicular tissue, as well as selection and enrichment strategies, and their outcome will be presented and discussed. While the literature revealed a plethora of mechanical dissociation/enzymatic digestion protocols, testicular tissue characteristics are often missing, precluding the comparison of protocols and their outcomes. Downstream selection and enrichment strategies showed promising results with flow cytometry reaching fractions with the highest purity. Future studies should focus on investigating digestion outcomes to elucidate potential influences on both the cell type-specific viability and the cell-to-cell interactions necessary for cell proliferation and differentiation of selected or enriched testicular cell types. Such research outputs will then also be crucial for further progress in in vitro spermatogenesis from testicular cell suspensions as another option for patients that banked testicular tissue at the time of a hematological cancer.