| Objective:To investigate the feasibility of rabbit adipose-derived stem cells (ADSCs) as the seed cell of tissue engineered skin after in vitro differentiation into epidermal stem cells by method of co-culture.Methods:1.Adipose was collected from the inguinal and neck region of 3～4 months old rabbits.Cells were isolated with adherent method and type I collagenase method and cultured in vitro.We identified ADSCs by observing their biological characteristics.2.HaCaT cells were cultured at the same time.3.Three groups:①co-cultured ADSCs for 3 days;②co-cultured ADSCs for 7 days;③P3 ADSCs.In this study we built up a co-culture system by transwell.ADSCs and HaCaT cells were separated with a polycarbonate membrane.Finally to investigate the morphology,and expression of CK19,Pan-CK and integrin-β1 on co-cultured ADSCs by immunocytochemistry.Results:1.Biological characteristics of ADSCs:①ADSCs had spindle and polygon shape adherent growth,and their growth and differentiation were active.②The growth curve was like "S" shape.The doubling time of ADSCs was about 22～25h.③Immunocytochemistry showed that ADSCs express CD29,CD44 but did not express CD 34 and CD45.2.Co-cultured ADSCs:①Co-cultured ADSCs got wider,and was like cosh shape which proved it was in immature phase.②Immunocytochemistry showed that co-cultured ADSCs express CK19, Pan-CK and integrin-β1.Co-cultured ADSCs contained ADSCs,ESCs and TA cells.Conclusion:1.The ADSCs were isolated with type I collagenase method and cultured in vitro.2.Rabbits' ADSCs differentiate to ESCs cultured with human HaCaT cells in vitro.3.The method,which built up a co-culture system by transwell,is highly efficient,no cross action,easy to practice,and economical. |
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