Following the Clinical Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University and written informed patient consent for tissue collection, MSCs were isolated following the protocol described in the previous study (23 (link)). In brief, fat tissues were isolated via liposuction from healthy patients defined as no history of malignancy or autoimmune deficiency. The fat tissues were shredded and digested with 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes at 37°C with gentle agitation. The cells were collected and cultured with MSC medium containing low glucose DMEM (Gibco) supplemented with FBS (10%, Gibco), fibroblast growth factor-basic (bFGF, 1 ng/ml, PeproTech) and penicillin/streptomycin (1%, Gibco).
When MSCs were approximately 90% confluent, they would be passaged with a split ratio of 1:3. MSCs of passage 2 were suspended in MSC medium with or without combinations of the following three small molecules, 2μM A-83-01 (MedChemExpress), 3μM CHIR99021 (Selleck), and 2μM Y-27632 (Selleck), and seeded on plates. The medium was changed every 2 days. From passage 5 to passage 7, the MSCs were cultured in MSC medium and used in subsequent analysis.
When MSCs were approximately 90% confluent, they would be passaged with a split ratio of 1:3. MSCs of passage 2 were suspended in MSC medium with or without combinations of the following three small molecules, 2μM A-83-01 (MedChemExpress), 3μM CHIR99021 (Selleck), and 2μM Y-27632 (Selleck), and seeded on plates. The medium was changed every 2 days. From passage 5 to passage 7, the MSCs were cultured in MSC medium and used in subsequent analysis.
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