Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (32): 5946-5952.doi: 10.3969/j.issn.2095-4344.2012.32.011

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Isolation, culture and biological characteristics of human adipose-derived stem cells in vitro

Tian Lin, Sun Xiao-Fang, Liu Hai-Bo, Luo Yu-Mei, Chen Xin-Jie , Huang Dong-Jian   

  1. Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • Received:2012-03-05 Revised:2012-04-27 Online:2012-08-05 Published:2012-08-05
  • Contact: Chen Xin-jie, M.D., Chief technician, Master’s supervisor, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China Co-corresponding author: Huang Dong-jian, M.D., Chief physician, Master’s supervisor, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • About author:Tian Lin★, Studying for master’s degree, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China doctor_tian118@yahoo.cn

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Abstract:

BACKGROUND: Human adipose-derived stem cells are widely used as seed cells in tissue engineering and regenerative medicine. Therefore, harvesting a large number of high-purity human adipose-derived stem cells is important for above-mentioned studies.
OBJECTIVE: To explore the suitable culture condition of human adipose-derived stem cells in vitro, and to enhance the proliferative ability of human adipose-derived stem cells.
METHODS: Collagenase I was used to digest and isolate human adipose-derived stem cells from intact fat of human abdomen. Human adipose-derived stem cells were purified using attachment method, cultured with low-glucose medium and passaged in vitro. Morphology was observed after Giemsa staining; the growth curve was drawn and cell cycle was analyzed. The karyotype of the passaged human adipose-derived stem cells was analyzed. Cells at the third passage were subjected to flow cytometry analysis, EdU incorporation and colony forming experiments.
RESULTS AND CONCLUSION: The morphology of primary cultured human adipose-derived stem cells was not the same, passaged human adipose-derived stem cells were spindle-shaped and arranged tightly in a vortex-like appearance. The growth curve was “S” shaped. Cell cycle analysis and EdU incorporation results showed that human adipose-derived stem cells cultured in vitro could maintain strong proliferative ability. Karyotype mapping showed that in vitro culture could not cause chromosome abnormalities of adipose-derived stem cells. Flow cytometry analysis showed that passage 3 adipose-derived stem cells were positive for CD29, CD44, CD90 and CD105, but they were negative for CD34 and CD45, with a clone formation rate of 8.8%. Under certain induction condition, adipose-derived stem cells could differentiate into adipocytes and osteoblasts. Human adipose-derived stem cells were successfully isolated by collagenase digestion method, cultured in vitro and showed strong proliferative ability.

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