Clinical grade cd271 microbead kit

Bone marrow-derived mononuclear cells (BM-MNC) were obtained using density gradient centrifugation over Lymphoprep (Axis-Sheld, Oslo, Norway). CD271⁺ BM-MNCs were isolated by immuno-magnetic positive selection using QuadroMACS system and clinical grade CD271 microbead kit (Miltenyi Biotec GmbH, Germany) following manufacturer’s instructions. The unselected and CD271-enriched BM-MNCs were then seeded into T25 culture flasks and 6 well culture plates respectively and cultured in good manufacturing practice (GMP) compliant MSC expansion medium containing 10% FCS (StemMACS, Miltenyi Biotec) at 37 °C in a 5% CO2 incubator. After 3 days, the non-adherent cells were discarded with the replacement of culture medium. The medium was changed twice weekly and cells were passaged to a new flask when the culture reached 80% confluence using standard Trypsin/EDTA (Sigma-Aldrich) treatment. The MSCs derived from BM-MNCs with or without CD271 enrichment were denoted as CD271- MSC and PA-MSC respectively. Each paired PA-MSC and CD271-MSC samples were generated from the same bone marrow donation, seeded at the same density (4 × 10³/cm²) and cultured/passaged under identical conditions. At each passage cell population doubling time was recorded. MSCs at passage 3 were used in all experiments throughout this work.