From Cow to Lab: Assessing Cytobrush Technique for Obtaining Endometrial Epithelial cells from Postpartum Cows for In-vitro Culture Studies

Sai Kumar B.A.A.¹; Mitzi Y.F. Vink^1,2; Kosala Rajapaksha³; Jaswant Singh⁴; Dinesh Dadarwal¹
1. Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada
2. Population Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
3. Agriculture and Agri-Food Canada, Saskatoon, Canada
4. Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada

Abstract Text: Bovine endometrial epithelial cells (BEEC) for in-vitro studies are typically obtained by dissection and enzymatic digestion of endometrial tissue from slaughterhouse-derived uteri. However, this lengthy and labor-intensive process can compromise cell viability and functionality. The cytobrush technique offers a rapid alternative to collect uterine cells from live cows for clinical assessment. This study was aimed to isolate BEEC from postpartum cows using the cytobrush method for in-vitro culture and to compare the effects of two proteolytic enzymes on the viability and apoptosis of BEEC. We hypothesized that BEEC collected from postpartum cows using cytobrush technique can be cultured in-vitro and that enzymatic digestion would negatively affect their viability. BEEC were harvested from dairy cows (n= 8, parity: 1-4, age: 2-5yr, five samples/cow) during early (12-15d) and late (40-44d) postpartum stages via the transvaginal route using modified triple-guarded cytobrush assemblies. Pooled samples (2.1 ml in DPBS with 1% antibiotic-antimycotic) from each cow were used to prepare pre-culture smears for phenotypic characterization using cell-specific cytoskeletal markers. Simultaneously, a portion (0.5ml) of pooled sample was cultured in DMEM-F12 with 10% FBS (37°C, 5% CO₂) until confluency, while the remainder (1.5ml, 250µl per treatment) underwent enzymatic digestion for 7.5- or 15-min with Collagenase I, Liberase^TM or EDTA. Post-digestion, cells were stained with Propidium Iodide (PI) and Alexa Fluor 488 conjugated Annexin V, and assessed for the cell viability and apoptosis, respectively, using flow cytometry. The in-vitro culture success rate between early and late postpartum stages was analyzed using a chi-square test. Additionally, two-way ANOVA was applied to analyze the effects of enzymatic type and digestion duration on cell viability and apoptosis. The mean (±SD) viable cell yield per cow was 1 x 10⁶ (±0.15 x 10⁶). Isolated BEEC expressed only cytokeratin, not vimentin, and exhibited swirling morphology up on in-vitro culture, reaching 85-100% confluency in 4-8d. Although not significant, fewer cows yielded culturable BEEC during early postpartum than the late postpartum stage (1/8 vs 5/8, p >0.1). BEEC treated with Liberase^TM had higher viability (p < 0.01) compared to those treated with EDTA during both early (93% vs 81%) and late (89% vs 63%) postpartum stages. Additionally, the percentage of non-apoptotic cells was higher (p < 0.02) in the Liberase^TM group (63%) than in the Collagenase I (41%) and EDTA (41%) groups during late postpartum period. Digestion duration did not significantly affect the proportions of live and non-apoptotic BEEC harvested during early and late postpartum stages (p >0.4). In conclusion, this study demonstrates that the cytobrush technique is an efficient, and minimally invasive method to collect BEEC from postpartum cows that are suitable for in-vitro culture. The findings confirm that enzymatic digestion negatively impacts BEEC viability, with Liberase™ being the most effective enzyme for maintaining cell viability while minimizing apoptosis. The technique described above would facilitate further research on BEEC characteristics and their responses to pathogens.