Stromal stem cells proliferate in vitro and may be differentiated along several lineages. The scarcity of stromal stem cells in tissues and the lack of reagents required obtaining freshly isolated cells of high purity and in quantities required for extensive analysis. In human bone marrow, for example, only 0.001–0.01% of nucleated cells form CFU-Fs. Adipose tissue is easy to obtain in large quantities and harbors a large number of cells with CFU-F ability; therefore, it should provide a readily available source of stromal stem cells in numbers sufficient to study their biology without having to resort to cell culture.
1. Adipose tissue was obtained by liposuction from abdominal, hip, and thigh regions of healthy female donors aged 18–39.
2. The stromal vascular fraction (SVF) was separated from adipose tissue using a procedure modified.
3. Lipoaspirate (300–400 ml) was washed with primary cell culture system containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) and 2.5 μg/ml amphotericin B.
4. Washed adipose tissue was digested for 2 h on a shaker at 37°C in primary cell isolation system.
5. Floating adipocytes were aspirated from pelleted SVF cells after centrifugation at 400 × g for 10 min.
6. Pellets were resuspended in red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH4Cl, pH 7.2) for 10 min at room temperature.
7. After resuspending SVF cells in primary cell culture system containing 2% fetal bovine serum (FBS), tissue clumps were allowed to settle for 1 min.
8. Suspended cells were passed through 100-μm and then 40-μm cell sieves.
9. Cell suspensions (15 ml) were applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes.
10. After centrifugation (400 × g, 30 min), cells at the gradient interface were collected, washed in HBSS, and passed through a 30-μm mesh.
11. Cell counts and viability assessment were performed.
References
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3. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J.D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147.
4. Andrew C. Boquest, Aboulghassem Shahdadfar, Katrine Frønsdal, Olafur Sigurjonsson, Siv H. Tunheim, Philippe Collas, and Jan E. Brinchmann. Isolation and Transcription Profiling of Purified Uncultured Human Stromal Stem Cells: Alteration of Gene Expression after In Vitro Cell Culture (2005). Mol. Biol. Cell 16: 1131-1141.
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