Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions.

This study explored the methodology for expanding equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood, and found that it is a feasible, time- and cost-efficient procedure for cellular therapy in equine medicine.

Methodology

• The researchers used mononuclear cells taken from 7 different samples of umbilical cord blood.
• Each sample was cryopreserved at a concentration of 1-2 × 10(9) cells per liter of cold freezing solution.
• The cells remained frozen for at least 6 months before the next step in the process, which was thawing.
• Thawing was done by placing the cryotubes that contained the cells in a 37°C water bath.

Results

• After being thawed, the average cell viability was measured at 98.7 ± 0.6%.
• In 6 of the 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 ± 2.6 days.
• The cell colonies reached 80% confluency within 12.3 ± 3.9 days.
• After undergoing three cell passages, the putative equine mesenchymal stromal cells were successfully immunophenotyped.
• This process involved using multicolour flow cytometry based on a selected 9 marker panel, and the results showed the cells were CD29, CD44 and CD90 positive and CD45, CD73, CD79α, CD105, MHC II and monocyte-marker negative.

Conclusion

This research presents a feasible time- and cost-efficient protocol for cryopreserving and then expanding equine mesenchymal stromal cells. This protocol could be beneficial for regenerative medicine in horses, as equine mesenchymal stromal cells have therapeutic potential. These cells, once expanded, can be used in cellular therapy to aid in the repair and regeneration of damaged tissues. This process, therefore, could be used as a practical approach in equine regenerative medicine.