# Transcriptomic Differences by RNA Sequencing for Evaluation of New Method for Long-Time In Vitro Culture of Cryopreserved Testicular Tissue for Oncologic Patients

## TL;DR

The study evaluates a new method for long-term in vitro culture of cryopreserved testicular tissue using RNA sequencing to assess transcriptomic differences.

## Contribution

A novel in vitro culture method using fibrin encapsulation and constant stirring is proposed to minimize thawing method impacts on testicular tissue.

## Key findings

- Fewest differentially expressed genes were observed between testicular tissue cultured after quick and slow thawing.
- Significantly up-regulated genes included C4B_2, LOC107987373, and GJA4, while SULT1A4, FBLN2, and CCN2 were down-regulated.
- Differential genes were enriched in pathways related to actin cytoskeleton regulation, lysosome function, and spermatogenesis.

## Abstract

Background: Earlier studies have established that culturing human ovarian tissue in a 3D system with a small amount of soluble Matrigel (a basement membrane protein) for 7 days in vitro increased gene fusion and alternative splicing events, cellular functions, and potentially impacted gene expression. However, this method was not suitable for in vitro culture of human testicular tissue. Objective: To test a new method for long-time in vitro culture of testicular fragments, thawed with two different regimes, with evaluation of transcriptomic differences by RNA sequencing. Methods: Testicular tissue samples were collected, cryopreserved (frozen and thawed), and evaluated immediately after thawing and following one week of in vitro culture. Before in vitro culture, tissue fragments were encapsulated in fibrin. Four experimental groups were formed. Group 1: tissue quickly thawed (in boiling water at 100 °C) and immediately evaluated. Group 2: tissue quickly thawed (in boiling water at 100 °C) and evaluated after one week of in vitro culture. Group 3: tissue slowly thawed (by a physiological temperature 37 °C) and immediately evaluated. Group 4: tissue slowly thawed (by a physiological temperature 37 °C) and evaluated after one week of in vitro culture. Results: There are the fewest differentially expressed genes in the comparison between Group 2 and Group 4. In this comparison, significantly up-regulated genes included C4B_2, LOC107987373, and GJA4, while significantly down-regulated genes included SULT1A4, FBLN2, and CCN2. Differential genes in cells of Group 2 were mainly enriched in KEGG: regulation of actin cytoskeleton, lysosome, proteoglycans in cancer, TGF-beta signaling pathway, focal adhesion, and endocytosis. These Group 2- genes were mainly enriched in GO: spermatogenesis, cilium movement, collagen fibril organization, cell differentiation, meiotic cell cycle, and flagellated spermatozoa motility. Conclusions: Encapsulation of testicular tissue in fibrin and long-time in vitro culture with constant stirring in a large volume of culture medium can reduce the impact of thawing methods on cryopreserved testicular tissue.