Refining the Protocol for Equine Umbilical-Cord-Derived Mesenchymal Stem Cell Isolation: A Pilot Study on Resource...

Equine mesenchymal stem cells (MSCs) are vital tools for preclinical studies and regenerative medicine, due to their multipotent nature and immunomodulatory potential. Wharton's jelly provides a non-invasive and abundant source of MSCs. However, the isolation from equine umbilical cord remains challenging, as tissue decontamination is difficult and the enzymatic isolation methods often yield variable amounts of cells. In addition, the established protocols are frequently labour-intensive, time-consuming, and costly. In this pilot study we adapted existing protocols with the aim to improve contamination rates, time commitment and costs. A thorough decontamination method and increased collagenase I digestion time from 3 h in the reference method (RM) to 24 h for the novel method (NM) were employed. Trilineage differentiation, RT-qPCR and flow cytometry were used to characterise both groups. Finally, a non-inferiority analysis was performed to confirm the feasibility of the NM. The changes in protocol led to the decrease in overtime and nightshifts, a faster postisolation expansion by reducing the culture duration by 13.5 ± 11 d, and minimised overall contamination rates (6.3% across 64 samples; RM = 3.1%, NM = 9.4%) compared with most published protocols. Consequently, we were able to reduce the costs per sample by 21 ± 21%.