Effects of cell dissociation treatment on early apoptosis and membrane integrity of in vivo collected bovine endometrial epithelial cells from postpartum cows.

We evaluated the effects of non-enzymatic and enzymatic dissociation agents on early apoptosis and membrane integrity of bovine endometrial epithelial cells (BEEC) harvested in vivo from postpartum cows via the cytobrush technique and assessed their potential for in vitro culture without enzymatic treatment. BEEC were collected from dairy cows (n = 8) at early (13 ± 1 days) and late (42 ± 2 days) postpartum stages using cytobrushes, with five samples per cow per time point. Pooled samples from each cow were used for phenotypic characterization and culture, or to assess treatment effects of EDTA, Collagenase I, and Liberase™ TM applied for 7.5 or 15 min. Early apoptosis and membrane integrity were evaluated using flow cytometry. Mean viable cell yield was 1 × 106 (±0.15 × 106) per cow. Cytosmears revealed large BEEC clusters expressing cytokeratin but not vimentin, and cultured cells exhibited swirling morphology, reaching 85-100% confluency within 4-8 days. Culture success was numerically higher in late versus early postpartum samples (5/8 vs 1/8), although not statistically significant. Treatment duration did not affect apoptosis or membrane integrity. Across stages, EDTA reduced live cell proportion compared to other treatments, while Liberase™ TM yielded the highest proportion of non-apoptotic cells (P < 0.05). The proportion of non-apoptotic cells was higher in early postpartum samples (P < 0.001). Overall, BEEC viability depended on dissociation method, with Liberase™ TM best preserving live cells. Primary BEEC collected via cytobrush can be cultured without enzymatic digestion, with enzymatic treatments applied later during passaging.