Impact of multiple isolation procedures on the differentiation potential of adipose derived canine mesenchymal stem cells

Objective: In regenerative biology, the most commonly used cells are adipose tissue-derived mesenchymal stem cells (AD-MSCs). This is due to the abundance and easy accessibility of AD-MSCs. Methods: In this study, canine AD-MSCs were harvested from different anatomical locations, i.e., subcutaneous (SC), omental (OM), and perirenal (PR). Various isolation techniques namely explants (TRT-I), collagenase-digestion (TRT-II), collagenasedigested explants (TRT-III), and trypsin-digested explants (TRT-IV) were used to segregate the MSCs to evaluate cell doubling time, viability, and adipogenic/osteogenic lineage differentiation potential. Results: The study showed that the SC stem cells had superior growth kinetics compared to other tissues, while the cells isolated through TRT-II performed better than the other cell isolation procedures. The metabolic status of cells isolated from dog adipose tissue indicated that all cells had adequate metabolic rates. However, SC-MSCs derived from TRT-III and TRT-IV outperformed those derived from TRT-I and TRT-II. The differentiation analysis revealed that cells differentiate into adipogenic and osteogenic lineage regardless of treatment, as demonstrated by positive oil red O (ORO) and Alizarin Red S (ALZ) stain. It is worth mentioning that cells derived from TRT-III had larger and more intracellular droplets compared to the other treatments. The TRT-I, -II, and -III showed greater osteogenic differentiation in cells isolated from PR and OM regions compared to SC-derived cells. However, the TRT-IV resulted in better osteogenic differentiation in cells from SC, followed by the OM and PR-derived cells. Conclusion: It is concluded that all methods of MSCs isolation from adipose tissues are successful; however, the TRT-II had the highest rate of cell re-assortment from the SC, while, TRT-II and -IV are most suitable for isolating cells from PR and OM adipose tissue.