Dynamic In Vitro 3D Culture of Cryopreserved Human Ovarian Cells: Transcriptomic Analysis by RNA Sequencing

Background: To enhance fertility, in vitro culture of ovarian tissue has become a commonly em-ployed approach before transplantation. As in vitro culture of ovarian tissue technology remains in the exploratory stage, developing a more effective 3D culture model is essential for optimizing follicle growth. Object: 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. Methods: Ovarian tissue samples were collected, cryopreserved (frozen and thawed), and cul-tured in vitro with 3D system for 7 days using TISSEEL Fibrin, followed by RNA sequencing and histological evaluation. 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). Results: KEGG analysis shows that in comparison to groups 1 and 3 (thawing ovarian tissue without culture), 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 endo-plasmic reticulum pathway and mainly down-related in the cell adhesion molecules pathway. Conclusion: Technology of the described dynamic in vitro 3D-cultivation of ovarian tissue for 7 days with TISSEEL Fibrin is informative and demonstrates that cryopreservation stimulates follic-ulogenesis and proliferative properties of the entire tissue.