Development of a cell culture system from bovine and bubaline endometrial cells recovered by a minimally invasive technique using a cytobrush

Ruminant endometrial cell culture is a common model system used to study uterine receptivity and related signaling in the maternal endometrium and pregnancy. The present study aimed to develop a minimally invasive technique by employing a cytobrush for collecting endometrial cell samples retrieved from cattle and buffalo and to establish and characterize the cells. The study was divided into two experiments. Experiment I evaluated the tissue invasiveness associated with different brush cytology rotations for 3, 5, 7, and 9 rounds compared with biopsy samples. In total, 30 areas of endometrial tissue samples from the uteri of six fresh, healthy, non-pregnant bovines from a slaughterhouse were examined histologically to determine tissue invasiveness based on the perpetual endometrial layer. The seven rounds of cytobrush rotation provided optimal results with detached epithelial cell lining and sloughed-off basement membrane while maintaining intact endometrial stroma, suggesting its suitability for subsequent cell culture. In Experiment II, primary endometrial cell cultures were established employing the 7-round brush cytology method from ten healthy, Holstein Friesian and swamp buffalo cows (n = 5, each). Endometrial cells were subcultured up to the third passage for further validation. Physical characterization utilized immunocytochemistry with pan-cytokeratin and vimentin co-staining. The cultured cells were functionally assessed by quantifying prostaglandin F2 alpha (PGF2α) secretion following 0 and 100 nM oxytocin challenge. Our findings showed that cytobrush sampling yielded sufficient seeding cells for culture. The proportions of endometrial epithelial cells relative to the stromal cells at passage 3 were 89.98 ± 7.21 % and 85.18 ± 2.66 % for cows and buffaloes, respectively. The secretion of PGF2α at 24 h increased significantly in bovine endometrial cell culture with 100 nM oxytocin (931.37 ± 292.69 pg/mL) compared with 0 nM oxytocin (194.06 ± 43.95 pg/mL; p = 0.026), as well as in bubaline endometrial cell culture, at 100 nM oxytocin (5.17 ± 1.54 pg/mL) compared 0 nM oxytocin (1.92 ± 0.76 pg/mL; p > 0.05). In conclusion, minimally tissue-invasive in vivo brush cytology sampling methods are effective for establishing a primary endometrial cell culture system in cows and buffaloes, providing a valuable model for studying reproductive physiology.