Cryopreservation of Testicular Cells and Tissues by Solid Surface Vitrification

Abstract

Cryopreservation of testicular cells and tissues may offer a potential alternative to fertility preservation in males wherein semen cryopreservation is not possible due to non availability of sperm (e.g. pre-pubertal males, non obstructive azoospermia etc.) or non-feasibility (e.g. posthumous reproduction, pre-meiotic barriers to spermatogenesis etc.). Testicular cells, such as Leydig cells, are also emerging as a new therapeutic modality for androgen replacement therapy. However, studies on cryopreservation of Leydig cells are scarce and, testicular cryopreservation protocols are sub-optimal in human as well as farm animals such as goats. This study investigated the cryopreservation of testicular cells by open [Solid Surface Vitrification (SSV) and Microdroplet (MD) method] and closed [Plastic Straw (PS) and Plastic Vial (PV)] vitrification, and applied the modified SSV for encapsulation vitrification. Mouse Leydig cell (TM3) line was initially used to avoid batch-to-batch variation, and the optimized protocol was tested for SSV of goat testicular cell suspension (TCS) and tissues. Effect of tissue size and organ culture methods for in vitro spermatogenesis were evaluated. Results showed that the SSV method was superior to MD, PS and PV methods of vitrification in terms of post-warming viability, cytoplasmic esterase enzyme activity, mitochondrial activity and cell growth (p<0.05). Vitrification induced the activity of reactive oxygen species (ROS), which could be annihilated by supplementation of 50 µM 2-mercaptoethanol (2-ME) or 100 µM glutathione (GSH) to improve the post-warming viability of the vitrified cells (p<0.05). The vitrified-warmed Leydig cells could completely regain their cell growth characteristics after 7 days of in vitro culture. The Leydig cells could also be encapsulated into alginate beads with encapsulation efficiency of 1.2 X 105 cells/ml per bead and could be vitrified successfully by SSV. However, the size of the beads significantly influenced the post-warming viability of cells (p<0.05). The optimized SSV was also applied to SSC-enriched goat TCS and testicular tissues. Results suggest that SSV could successfully cryopreserve DAZL- and PGP9.5- positive putative SSCs, which could proliferate in vitro and form cell colonies. The SSV vitrified-warmed TCS showed similar mitochondrial activity, rate of cell proliferation and population doubling time of total cells comparable to non-vitrified control. However, during in vitro culture, vitrified-warmed cells showed significantly lower mitochondrial activity than those of non-vitrified controls, which was associated with increased ROS activity. The optimized SSV method could also successfully vitrify goat testicular tissue. However, the post-warming viability varied with the tissue size: small size tissue (~4 mm3) resulted in better viability than medium (~9 mm3) or large (~16 mm3) sized testicular tissue. The low viability of vitrified-warmed testicular tissue was associated with increased ROS activity, tissue fracture and damage to both histo-architecture and cellular components of the testis. It was also observed that organ culture (OC) method was superior (p<0.05) to hanging drop (HD) method in terms of post-warming metabolic activity on Day 7 and Day 14 of culture, respectively. Histology and scanning electron microscopy (SEM) showed the rupture of basal membrane, surface morphology and cell loss due to vitrification. However, histology and immunohistochemistry showed the progression of in vitro spermatogenesis and formation of elongated spermatozoa in both fresh and vitrified-warmed testis tissue cultured by OC method. In conclusion, results of this study suggest that modified SSV may offer a viable method for vitrifying single cell suspension of Leydig cells, mixed population of goat TCS as well as goat testicular tissue. Future work should determine the ability of vitrified-warmed testicular cells and tissues to restore male fertility upon their transplantation in recipient testes of animal models.