Isolation, Culture In Vitro And Identification Of Human Adipose-derived Stem Cells

Objective:The purpose of this research is to isolate, culture in vitro and identificate human ASCs, to observe biologic characters and generation potency of ASCs, and establish substantial foundation for deeper research of tissue engineering.Methods:Trivial adipose tissue is placed in HG DMEM medium with equal volume of 0.075% collagenase I digestion solution.The mixture is placed in the 37℃, 5%CO2 incubator for 24 hours, then transferred into a centrifuge tube, centrifuged. The cell suspension is aspirated and transferred into a cell culture plate with 6 holes, and incubated at 37℃, 5%CO2.The medium is changed twice per week. We choose the third generation cells for immunohistochemistry stain, then obseve them under light microscope. We add osteogenic and adipogenic induction medium into the third generation cells separately . Control group is set up with contrast medium only. The test of AKP content is carried out 72 hours later . Alizarin red stain is carried out 8 days later. Oil-red O stain is carried out 2 weeks later.Results:A few major ancipital cells start to adhere 24 hours after inoculation, most cells start to adhere 48 hours later, with fusifore shape and bulky ecphyma. After 5 to 7 days, cells disintegrate gradually, and fuse into monolayer. The cellular shape become fusiform after passaging, with the nucleus in the middle, mostly with only one nucleolus, round or orbicular-ovate.Immunohistochemistry stain shows that CD44 and CD49d are positive, CD45 and CD106 are negative.Oil-red O stain shows that the induction group is positive, and the control group is negative 2 weeks after adipogenic induction.The test of AKP content in the induction group inceases obviously compared with the control group 72 hours after osteogenic induction.Alizarin red stain shows that the induction group is positive, and the control group is negative 8 days after osteogenic induction.Conclusion:We can isolate fibroblast-like cells from human subcutaneous adipose tissue, culture and passage them in vitro using primary cell culture technique. These cells can differentiate into adipocytes or osteocytes in induction mediums. Initial identification demonstrates that these cells are ASCs.