Optimization of In vitro Culture Conditions of Interleukin-10 Secreting B Regulatory Cells of Human Origin

AbstractAim: B-regulatory cells (B-regs), commonly characterized as CD19+/38hi/24hi, comprise of a major subset of IL-10 secreting B-cells. We report optimization of culture conditions for B-regs of human origin, obtained from in-vitro co-culture of donor adipose tissue derived mesenchymal stem cells (AD-MSC) and renal allograft recipient (RAR) peripheral blood mononuclear cells (PBMC) for potential cell therapy.Material and methodology: Five sets of 6-well plates were filled with proliferation media. Each plate was seeded with 5 × 104 ADMSC and 1 × 106 cells of responder and stimulator peripheral blood mononuclear cells (PBMC) and incubated at 37˚C, 5% CO2 after addition of lipopolysaccharide–E.coli-K12 strain (LPSEK) in varying concentrations. At each time point, one plate was withdrawn and contents centrifuged. IL-10 concentration of cell supernatant was estimated by quantitative ELISA. Pellet was resuspended to determine morphology, sterility, total count, viability and immunophenotype of cells.Results: The highest B-reg population (0.19×106 cells/ml) was noted at 24 hours with 400 ng/ml LPS-EK, which also correlated with optimum secretion of IL-10 (1.58 pg/ml), for the same time interval.Conclusion: The present study provides valuable information about the dynamics of IL-10 secretion by in vitro generated human B-regs, obtained from donor AD-MSC and recipient PBMC.