# Dynamic in vitro 3D culture of cryopreserved human ovarian tissue: transcriptomic analysis by RNA sequencing

## TL;DR

This study explores how 3D in vitro culture of thawed human ovarian tissue affects gene expression, finding that it promotes follicle development and alters key biological pathways.

## Contribution

The study introduces a dynamic 3D culture method using TISSEEL Fibrin for thawed ovarian tissue and identifies specific transcriptomic changes linked to follicular growth.

## Key findings

- 3D culture with TISSEEL Fibrin promotes follicle cell migration and development.
- RNA sequencing shows upregulation of lysosome and endoplasmic reticulum pathways in cultured tissue.
- 3D culture weakens cell adhesion and causes minimal cell damage compared to non-cultured thawed tissue.

## Abstract

To explore the in vitro 3D culture of ovarian tissue thawed in two different ways with TISSEEL Fibrin and assessed transcriptome differences by RNA sequencing.

Human ovarian tissue samples were collected, and the fragments used for the experiments were obtained from tumor patients involved in a fertility treatment program. After cryopreservation (frozen and thawed after initial cryopreservation), with or without 3D culture with TISSEEL Fibrin. The culture flask is agitated at 75 times per minute using a rotary shaker during the entire culture process. Four experimental groups were formed. Frozen tissue after quick thawing at 100 °C (Group 1), frozen tissue after quick thawing at 100 °C and in vitro 3D culture for 7 days with TISSEEL Fibrin (Group 2), frozen tissue after slow thawing at 37 °C (Group 3), frozen tissue after slow thawing at 37 °C and in vitro 3D culture for 7 days with TISSEEL Fibrin (Group 4). All groups were followed by RNA sequencing and histological evaluation.

Hematoxylin-eosin (HE) staining has shown that the follicle cells have the tendency to develop and actively migrate into the fiber. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis shows that in comparison to groups 1 and 3 (thawing ovarian tissue without culture), Differential Expression Genes (DEGs) in groups 2 and 4 (thawing ovarian tissue with in vitro 3D culture), are mainly enriched and up-related in the lysosome pathway and protein processing in the endoplasmic reticulum pathway and mainly down-related in the cell adhesion molecules pathway.

The technology of the described dynamic in vitro 3D-cultivation of ovarian tissue for 7 days with TISSEEL Fibrin is informative and demonstrates that it may promote follicular growth, actively migrate into the fiber, weaken cell adhesion, and cause little cell damage.