Cytotoxicity Assay of MSC-PBMC Co-culture

Proliferating and confluent MSC544 populations were seeded in 24-well plates (Nunc, Wiesbaden, Germany) at a density of 1 × 10⁵ cells/cm² and allowed to adhere, respectively. Peripheral blood mononuclear cells (PBMCs) were prepared from peripheral blood by Ficoll density gradient centrifugation as described elsewhere [44 (link)]. Unstimulated PBMCs were added and the MSC populations in direct contact at a ratio 10:1 in RPMI 1640 media (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1mM L-glutamine, 50 U/mL penicillin, 0.5mM sodium pyruvate and 50 μg/mL streptomycin (R10) [45 (link)]. Following the co-culture, PBMCs as effector cells were harvested and incubated with ⁵¹Cr–labeled K652 target cells at different effector to target (E:T) ratios in R10 for four hours. Samples were centrifuged (300g /5 min) and released radioactivity (counts per minute) was determined from cell-free supernatants [46 (link)].