Adipose mesenchymal stem cells (AMSCs) were isolated from adipose tissue obtained by lipoaspirates. A total number of six lipoaspirate samples were collected after informed written consent of donors. Ethical approval was obtained from the Medical Research Ethics Committee of the Centro di Riferimento Oncologico, IRCCS, Aviano, Italy (Consent CRO-2016-30).
Lipoaspirates were enzymatically dissociated using a 0.05% collagenase II solution for 20 minutes at 37 °C (Worthington) and, after neutralization of the enzyme, were centrifuged at 500 × g for 5 minutes and filtered through a 70 μm nylon mesh (Merck Millipore). Cells were seeded in minimum essential medium-α (MEM-α) supplemented with 10% FBS (Gibco), penicillin/streptomycin solution (10 mL/L), alanine/glutamine solution (2 mM), human epidermal growth factor (10 ng/ml), insulin solution (10 μg/ml), 2-fosfo-L-ascorbic acid, trisodium salt (100 μM) and dexamethasone (0.01 μM) (all from Sigma-Aldrich). Culture were kept at 37 °C, 5% CO2 and 95% humidity and cells were characterized by flow cytometry using MSCs positive markers (CD29, CD73, CD90 and CD105) and hematopoietic negative markers (CD34 and CD45) as described previously42 (link). Cells were used for experiment between passage 2 and 5.
All methods of analysis were performed in accordance with the relevant guidelines and regulations with appropriate quality control.
Lipoaspirates were enzymatically dissociated using a 0.05% collagenase II solution for 20 minutes at 37 °C (Worthington) and, after neutralization of the enzyme, were centrifuged at 500 × g for 5 minutes and filtered through a 70 μm nylon mesh (Merck Millipore). Cells were seeded in minimum essential medium-α (MEM-α) supplemented with 10% FBS (Gibco), penicillin/streptomycin solution (10 mL/L), alanine/glutamine solution (2 mM), human epidermal growth factor (10 ng/ml), insulin solution (10 μg/ml), 2-fosfo-L-ascorbic acid, trisodium salt (100 μM) and dexamethasone (0.01 μM) (all from Sigma-Aldrich). Culture were kept at 37 °C, 5% CO2 and 95% humidity and cells were characterized by flow cytometry using MSCs positive markers (CD29, CD73, CD90 and CD105) and hematopoietic negative markers (CD34 and CD45) as described previously42 (link). Cells were used for experiment between passage 2 and 5.
All methods of analysis were performed in accordance with the relevant guidelines and regulations with appropriate quality control.
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