Stromal Vascular Fraction From Canine Adipose Tissue Comprises Mesenchymal Stromal Cell Subpopulations Characterized by Time-dependent Binding Affinity to Culture Plastic

Abstract

BACKGROUD: Mesenchymal Stromal Cells (MSC) from adipose tissue are among the most extensively investigated cells for clinical applications both in human and veterinary medicine. Their isolation is mostly carried out by collagenase digestion followed by filtration and seeding. Non-adherent cells are then removed from culture 48 hours upon seeding. Our hypothesis is that fragments eliminated by filtration or discarded after 48h might contain residual MSC that adhere later to the plastic, also after 6 days (144 hours) upon isolation. The aim of this study is to evaluate the basic features of cells that adhere to the plastic at 3 different time points after isolation in order to speculate on the possible existence of MSC subpopulations that are located in different tissue niches.METHODS: Subcutaneous adipose tissue collected from 3 dogs was minced and digested with collagenase I. Three cell populations were obtained at different time points from isolation (48h, 96h, and 144h). They were expanded until passage 3 and characterized by flow cytometry for positive (CD90, CD44, CD29) and negative (CD14, MHC2, CD45) MSC markers as well as for CD31 (endothelial cell marker), CD146 (pericyte marker), alpha-SMA (smooth muscle cell marker). At passage 3, cells were evaluated for viability (MTT assay), doubling time, and trilineage differentiation ability.RESULTS: No significant differences between the 3 subpopulations were observed. They were all characterized by the expression of MSC positive markers and by the absence of negative markers. CD31, CD146, and alpha-SMA were expressed by less than 5% of the cells in all the 3 sub-populations. No differences in differentiation ability and viability were detected. Doubling time ranged between 25 and 30 hours in all the 3 experimental groups.CONCLUSIONS: The 3 cell subpopulations were similar in terms of immunophenotype, proliferation, and differentiation potential. In this view, the procedure of sequential adhesions seems to be a useful method to efficiently improve MSC yield. Indeed, it allows for optimized cell recovery, reducing the amount of sampled tissue and shortening the time necessary to obtain an adequate number of cells for clinical applications. However, functional differences cannot be excluded, and potency assays are required in order to explore possible distinct biological attitudes.