Isolation of equine bone marrow-derived mesenchymal stem cells: a comparison between three protocols

Area of Science:

• Veterinary Medicine
• Stem Cell Biology
• Biotechnology

Background:

• Standardization of equine bone marrow (BM) mesenchymal stem cell (MSC) isolation is crucial for comparing therapeutic trial outcomes.
• Current protocols for equine MSC isolation lack direct comparison, hindering inter-site data validation.

Purpose of the Study:

• To compare three distinct equine BM MSC isolation protocols: classic plastic adherence, Percoll gradient density separation, and Ficoll gradient density separation.
• To evaluate cell viability, MSC yield, post-culture cell numbers, and functional characteristics (self-renewal and multilineage differentiation) for each protocol.

Main Methods:

• Equine BM aspirates were collected from six mares.
• MSCs were isolated using three methods: classic plastic adherence, Percoll, and Ficoll gradient density separation.
• Key metrics assessed included cell viability, MSC yield, cell expansion after 14 days, colony-forming unit (CFU) assays for self-renewal, and multilineage differentiation capacity.

Main Results:

• The Percoll protocol yielded significantly higher MSCs (6.8 ± 3.8%) compared to the Classic protocol (1.3 ± 0.7%).
• Post-culture cell numbers were highest for Percoll (24.0 ± 12.1 x 10^6), followed by Ficoll (14.6 ± 9.5 x 10^6) and Classic (4.1 ± 2.5 x 10^6) per 10 ml BM.
• No significant differences were observed in cell viability or osteogenic/chondrogenic differentiation across the protocols.

Conclusions:

• The Percoll gradient density separation protocol demonstrated superior MSC yield and self-renewal potential at initial passage.
• While Ficoll showed lower self-renewal at Passage 0, all protocols yielded equivalent results by Passage 1.
• The Percoll protocol is recommended for equine MSC isolation due to its efficiency and potential to minimize culture time.