Bovine Genital Mucosa Cultivation Protocol

From three cows in the luteal phase and three cows in the follicular phase, posterior vagina, cervix and uterus body were collected at the local abattoir immediately after slaughter. The cultivation protocol of bovine genital mucosa was performed as described before [22 (link)–24 (link)]. In brief, genital tissues of cows were immediately placed in phosphate buffered saline (PBS), supplemented with 1000 U/mL penicillin (Continental Pharma, Puurs, Belgium), 1 mg/mL streptomycin (Certa, Braine l’Alleud, Belgium), 1 μg/mL gentamycin (Invitrogen, Paisley, UK) and 5 μg/mL fungizone (Bristol-Myers Squibb, New York, USA) on ice for transportation to the laboratory. The mucosae from vagina, cervix and uterus body were stripped from the underlying layers. Afterwards, tissues were cut into small equal square pieces (on average 25 mm²). Finally, genital mucosa was placed on sterilized gauzes in 6-well plates for culture. The explants were cultured in serum-free medium [50% DMEM (Invitrogen)/50% Ham’s F-12 Gluta-MAX (Invitrogen)], supplemented with 100 U/mL penicillin (Continental Pharma), 0.1 mg/mL streptomycin (Certa) and 1 μg/mL gentamycin (Invitrogen) for up to 96 h (37 °C and 5% CO₂).