Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (14): 2601-2606.doi: 10.3969/j.issn.1673-8225.2010.14.027

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Isolation, culture and multi-directional induced differentiation of rabbit adipose derived stromal stem cells  

Yang Hao, Wu Di, Li Shi-he, Zhu Xiao-song   

  1. Department of Orthopaedics, First Affiliated Hospital, Kunming Medical University, Kunming   650032, Yunnan Province, China
  • Online:2010-04-02 Published:2010-04-02
  • Contact: Wu Di, Doctor, Attending physician, Department of Orthopaedics, First Affiliated Hospital, Kunming Medical University, Kunming 650032, Yunnan Province, China xywudi@sohu.com
  • About author:Yang Hao★, Master, Attending physician, Department of Orthopaedics, First Affiliated Hospital, Kunming Medical University, Kunming 650032, Yunnan Province, China chariotrome@yahoo.com.cn
  • Supported by:

    the International Cooperation, i.e., the Special Project of Vitalizing Trade with Science and Technology of Department of Science and Technology of Yunnan Province in 2006, No. 2006GH18*

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Abstract:

BACKGROUND: Subcutaneous fat of human body is a rich reservoir of adipose derived stromal stem cells (ADSCs). ADSCs can proliferate rapidly when being cultured in vitro, and has the capacity of multi-directional differentiation. ADSCs attracted much attention in research of tissue engineered seed cells.
OBJECTIVE: To isolate and culture stromal vascular fraction (SVF) cells from rabbit subcutaneous fat in vitro, and to testify whether it has multiple differentiation capacity. 
METHODS: SVF cells were isolated in vitro from rabbits, and cultured under standard condition. Cellular surface antigens CD44, CD45 and CD29 of passage 3 SVF cells were examined using flow cytometry. Passage 3 SVF cells were induced to differentiate into osteoblasts, chondrocytes, and lipocytes. Oil red staining was used to examine lipocyte induction. Alkaline phosphatase (ALP) staining, alizarin red staining and von Kossa staining were used to examine osteoblast induction. Type Ⅱ collagen immunohistochemical staining and type Ⅱ collagen mRNA RT-PCR were used to examine chondrocyte differentiation.
RESULTS AND CONCLUSION: Primary SVF cells were multi-angular or short spindle-shaped. Passage 3 SVF cells were long spindle-shaped. Flow cytometry showed CD44+ , CD29+ , CD45- . Oil red staining exhibited positive reaction in lipocyte induction group. ALP staining, alizarin red staining and Von kossa staining demonstrated positive reactions in osteoblast induction group. Type Ⅱ collagen immunohistochemical staining and alcian blue staining have suggested positive reactions at 14 days of chondrogenic induction group. RT-PCR of type Ⅱ collagen mRNA test showed that the product band had strong signal at 14 days of chondrogenic induction group compared with that before induction. Above mentioned results have indicated that SVF cells isolated from rabbit subcutaneous fat have identical surface makers of stem cells, and have the ability to differentiate into lipocytes, osteoblasts and chondrocytes in vitro by induction, and it could be concluded that the SVF cells were ADSCs.

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