Isolation, Culture, Characterization, and Differences of Adipose-DerivedMesenchymal Stromal Cells from Male and Female Kunming Mice
LIU Yuxuan, LI Jingyang, DONG Hengyuan, XIA Mingyang, ZHANG Li*
This study investigates the methods for isolating, culturing, and characterizing AD-MSCs(adipose-derived mesenchymal stromal cells) from Kunming mice, as well as differences in their biological characteristics between male and female mice. In this study, AD-MSCs from Kunming mice were isolated using anenzymatic digestion method under sterile conditions for primary culture. Cell morphology was observed, and P4,P8, P12, P16 cell growth curves were analyzed. Immunofluorescence and flow cytometry were employed to detectthe expression of cell surface markers (CD29, CD31, CD34, CD44, CD45, CD90), assessed their multipotent differentiation capacity through adipocytes, osteocytes, and chondrocytes induction, while simultaneously quantifyingthe induction positivity rate, and evaluated cellular senescence.This study successfully isolated AD-MSCs exhibiting spindle-shaped, fusiform, or polygonal morphology during adherent growth, cell growth curves displaying atypical “S” shape; AD-MSCs-F (adipose-derived mesenchymal stromal cells from female Kunming mice) and ADMSCs-M (adipose-derived mesenchymal stromal cells from male Kunming mice) at P4, P8, and P16 all exhibitedpositive expression of CD29, CD44, and CD90, while CD31 and CD45 were largely undetectable, the expression ofCD34 varied by sex, as follows: CD34 expression in AD-MSCs-F increased with passage number to 53.6%, whileAD-MSCs-M remained negative; prior to P12, AD-MSCs-F proliferated more rapidly than AD-MSCs-M; after P12,their proliferation slowed, and senescence set in earlier, with more severe senescence observed at P16. Under adipogenic and osteogenic induction, the percentage of oil red O and alizarin red-positive areas was higher in AD-MSCsF than in AD-MSCs-M. There was no significant difference in the percentage of alcine blue-positive area underchondrogenic induction. This study successfully established an in vitro culture system and characterization methodsuitable for Kunming mouse AD-MSCs, successfully isolated and characterized Kunming mouse AD-MSCs. Female and male mouse AD-MSCs are similar in morphology and basic cell characteristics, however, there are certaingender differences in proliferative capacity, differentiation capacity, aging, and CD34 expression. This provides differentiated experimental evidence and technical support for AD-MSCs research in basic science, tissue engineering,and regenerative medicine.
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