70 μm nylon mesh

Infrapatellar fat pad in the knee joint cavity of New Zealand white rabbits (1 month) was extracted according as established previously [14 (link)]. In brief, fat were cut into small pieces (~ 2 mm) and enzymatically dissociated using a 0.05% collagenase II solution (Worthington, Columbus, OH, USA) for 30 min at 37 °C. After neutralization of the enzyme, cells were centrifuged at 500×g for 5 min and filtered through a 70-μm nylon mesh (Merck Millipore, Danvers, MA). ADSCs at passage 3 were used. ADSCs were characterized using flow cytometry with stem cells markers: CD105, CD45, CD73 and CD34. Moreover, ADSCs were characterized based on their adipogenic, osteogenic, and chondrogenic capacities.

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 previously^{42 (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.