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. 2023 Mar 27;13(7):1175.
doi: 10.3390/ani13071175.

The Stromal Vascular Fraction from Canine Adipose Tissue Contains Mesenchymal Stromal Cell Subpopulations That Show Time-Dependent Adhesion to Cell Culture Plastic Vessels

Gabriele Scattini  1 , Martina Pellegrini  2 , Giulio Severi  2 , Monica Cagiola  2 , Luisa Pascucci  1
Affiliations

The Stromal Vascular Fraction from Canine Adipose Tissue Contains Mesenchymal Stromal Cell Subpopulations That Show Time-Dependent Adhesion to Cell Culture Plastic Vessels

Gabriele Scattini et al. Animals (Basel). .

Abstract

Adipose-derived mesenchymal stromal cells (MSCs) are extensively studied in both human and veterinary medicine. Their isolation is usually performed by collagenase digestion followed by filtration and removal of nonadherent tissue remnants 48 h after seeding. We observed that waste tissue fragments contain cells that adhere belatedly to the plastic. We aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Adipose tissue from three dogs was enzymatically digested. Three cell populations that adhered to the culture plastic 48, 96, and 144 h after seeding were obtained. After expansion, they were analyzed by flow cytometry for MSC-positive (CD90, CD44, and CD29) and -negative (CD14, MHCII, and CD45) markers as well as for endothelial, pericyte, and smooth muscle cell markers (CD31, CD146, and alpha-SMA). Furthermore, cells were assessed for viability, doubling time, and trilineage differentiation ability. No significant differences were found between the three subpopulations. As a result, this procedure has proven to be a valuable method for dramatically improving MSCs yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Further studies are needed to uncover possible different functional properties.

Keywords: adipose tissue; cell culture; dog; immunophenotype; mesenchymal stromal/stem cells; stromal vascular fraction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Cumulative cell number of the three populations. Average number of cells obtained for the three populations (Ad1, Ad2, and Ad3) at different passages from five grams of dog adipose tissue. Mean values of the three dogs. (B) Doubling time of the three populations. Comparison of doubling of the three populations (Ad1, Ad2, and Ad3). Histograms represent the mean value calculated from three different dogs.
Figure 2
Figure 2
Multilineage differentiation. Representative images of trilineage differentiation induced on the 3 cell populations under study. White scale bars, 50 µm; Black scale bars, 200 µm. RT-PCR products for tissue-specific mRNA; FABP4 and PPARG2 for adipogenic differentiation; OPN and OSX for osteogenic differentiation; ACAN and COL2A1 for chondrogenic differentiation.
Figure 3
Figure 3
Relative viability of the three populations. Viability of the three populations (Ad1, Ad2, and Ad3) was measured with MTT assay. Absorbance values were normalized for the AD1 value. Histograms represent the mean relative viability values calculated for the three donor dogs.
Figure 4
Figure 4
Immunophenotype profiles. Representative histograms of positive cells for MSC negative markers (A), positive markers (B), and other stromal markers (C). Mean values were calculated for the three dogs.
See this image and copyright information in PMC

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