Ex vivo culture,identification and differentiation of human adipose derived stem/stromal cells

Objective To observe morphology,identify cell surface CD marker and test the ability of proliferation and differentiation of adipose derived stem/stromal cells(ASCs) which were isolated from intact fat ex vivo.Methods ASCs were isolated by collagenase digesting from intact fat of abdomen harvesting of skin grafts.The 3rd passage ASCs were applied to do the experiments: HE staining,flow cytometry,MTT method and cell cycle testing.Besides,passage 3 cells were induced to differentiate into lipocytes and osteocytes.Oil red staining was applied to examine adipogenic induction.Alizarin red staining was applied to examine osteogenic induction.Results Primary ASCs were multi-angular and short spindle cells at the first culture fluid changing.HE staining showed that Passage 3 cells grew like swirls and were long spindle-shaped.Flow cytometry showed CD29 +,CD31-,CD34-,CD44 +,CD45-,CD49 +,CD106-,CD133-.MTT manifested the growth and proliferation of ASCs was great.Cell cycle testing showed ASCs in G1 period accounted for 86.8% and G2 period 8.77%.Oil red staining and alizarin red staining both demonstrated positive reactions but not in control groups.Conclusion Human ASCs were a kind of mesenchymal stem cells with great application prospect,which characterized adherent growth,multipotent differentiation and stem cell phenotype.