【Title】

“Optimization of isolation and culture conditions of highly homogenous human adipose-derived mesenchymal stem cells”living pictures

【Source】

Journal of Clinical Rehabilitative Tissue Engineering Research  November 5, 2009  Vol.13 No. 45，8861-8864

【Author】

Yang Xu-fang^1,2, He Xu¹, Zhang Li-hong¹, Sun Mei¹, Xu Hong¹, Gao Yun-he¹, Li Yu-lin¹

【Setting】

¹Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun  130021, Jilin Province, China; ²Laboratory of Physiology, Mudanjiang Medical University, Mudanjiang  157001, Heilongjiang Province, China

【Found】

Supported by: the National High Technology Research and Development Program of China(“863” Program), No.2004AA205020*; National Natural Science Foundation of China, No.30700872*; Science Foundation for the Excellent Youth Scholars of Jilin University, No.419070100050*; the Medical Scientific Research Foundation of Health Department of Jilin Province*

【Abstract】

BACKGROUND: Up to date, there is not an acceptable method for isolating, culture and amplifying human adipose-derived mesenchymal stem cells (hADSCs).

OBJECTIVE: To explore the most effective way to obtain highly homogenous and undifferentiated hADSCs.

DESIGN, TIME AND SETTING: The in vitro cytology experiment was performed at the Key Laboratory of Pathobiology, Ministry of Education, Jilin University from June to December 2008.

MATERIALS: Human abdominal adipose tissue resected in the surgery was supplied by the Affiliated Hospital of Jilin University. The informed consent was obtained from patients.

METHODS: Human adipose tissue was removed connective tissue and blood vessel, followed by incubation in 0.1% type Ⅰ collagenase for 60 minutes at 37 ℃, filtrated then centrifuged. Consequently, the subnatant precipitation was cultured with LG-DMEM containing 10% fetal bovine serum, incubated at 6-well plate with density of 1×10⁹/L, and placed in incubator of 5% CO₂ at 37 ℃. The cultured cells were passaged when the cells reached 80%-90% confluency, and the 3^rd passage of cells were induced to osteogenic and adipogenic differentiation.

MAIN OUTCOME MEASURES: Morphological characteristics of hADSCs were observed by laser scanning confocal microscope. Immunophenotypes, cell cycle and growth curve of hADSCs were detected by flow cytometry and immunofluorescent techniques. In addition, the multiple differentiation potential of hADSCs was detected.

RESULTS: hADSCs presented fibroblast-like morphological feature with a flocked array. The 3^rd passage of hADSCs had unique immunophenotypes and they were positive for CD73, CD44, CD166, CD105 and CD29, but negative for CD31, CD34, CD45 and HLA-DR. Cell cycle result showed that they had the typical growth characteristics of stem cells, namely, 83.81% cells stayed at G₀/G₁ stage, only 16.19% cells were stayed at S+G₂/M stage; The latent phase of the primary culture cells was 2 days prior to and after incubation, followed by 3-6 days of logarithmical proliferation, reached a peak at day 6, and entering the growth platform phase with lower growth speed. The alkaline phosphatase was positive expressed at week 2 of osteogenic induction. And the positive expression of oil-O red staining could be seen at day 3 of adipogenic induction.

CONCLUSION: Cells contamination can be reduced by removing connective tissue and blood vessel of adipose tissue, and 0.1% type Ⅰ collagenase for 60 minutes can effective separated stroma cell to matrix fiber, furthermore, ensure a sufficient contact between collagenase and tissures, which provide an supportive for harvesting highly homogenous hADSCs.

【Number of pictures】

4 figures

Figure 1  Morphological features of human adipose-derived mesenchymal stem cells (hADSCs)

Figure 2  Immunofluorescence of 3^rd passage of human adipose-derived mesenchymal stem cells (×200)

Figure 3  Positive expression of alkaline phosphatase at 2 weeks of osteogenic induction  (×200)

Figure 4  Positive expression of oil-O red staining at day 3 of adipogenic induction (×200)

【Full text】

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