Endometrial tissue cryopreservation and organoid culture from domestic and endangered equids

Three studies were performed to evaluate the cryo-sensitivity and culture conditions required to establish equine endometrial organoid cultures in vitro. The endometrium, the inner lining of the uterus, responds to hormonal stimuli throughout the estrous cycle and is in direct contact with semen during breeding and with the conceptus during pregnancy. Experiment 1 evaluated three concentrations (0%, 10%, and 20% v/v) of dimethyl sulfoxide (DMSO) as a cryoprotectant during cryopreservation (slow freezing) of domestic mare endometrial explants. Both 10% and 20% DMSO were suitable concentrations of cryoprotectant for maintaining cellular viability, structural integrity, and gene expression of the tissues. Then, a 5-day explant culture utilizing a fluid-gas interface was performed on fresh and frozen-thawed endometrial explants comparing low and high glucose in culture media. This was the first report of extending equine endometrial explant cultures successfully beyond 48 hours, though a central core of necrosis, common in explant cultures, occurred. There were no differences in viability, structural integrity, or gene expression between low and high glucose in the culture media. For Experiment 2, the same study design as Experiment 1 was utilized for endometrial explants from Persian onagers, an endangered wild ass native to Asia. Only the 10% and 20% DMSO concentrations were evaluated as 0% was unsuccessful in domestic mares. Again, both 10% and 20% DMSO were equally suitable as cryoprotectants for endometrial explants, and glucose concentration did not affect the endometrial explants during 5-day explant culture. Comparison of fresh and frozen-thawed tissue derived endometrial organoids, three-dimensional cell cultures, were then assessed in Experiment 3 for endometrial tissue from both domestic and Przewalski’s horses, an endangered equid native to Asia. This was the first report of endometrial organoids derived from a domestic or wildlife species, as they have only been previously reported in mice and women. These endometrial organoids were maintained long-term (24 days) and responded with gene expression changes in response to exogenous hormone stimulation, the first in vitro cell cultures in mares able to accomplish these goals.