Cell Biology: Research & TherapyISSN: 2324-9293

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Research Article, Cell Biol Henderson Nv Vol: 7 Issue: 1

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

Gupte KS1, Vanikar AV1,2*, Patel CN2, Bhargav AG2, Patel JV2 and Thakkar UG1,2

1Department of Regenerative Medicine and Cell Therapy, Transplantation Biology Research Lab, G.R. Doshi and K.M. Mehta Institute of Kidney Diseases & Research Centre (IKDRC)- Dr. H.L. Trivedi Institute of Transplantation Sciences (ITS), India

2Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology. G.R. Doshi and K.M. Mehta Institute of Kidney Diseases and Research Centre (IKDRC) - Dr. H.L. Trivedi Institute of Transplantation Sciences (ITS), India

*Corresponding Author : Dr. Aruna V. Vanikar, MD, PhD
Professor and HOD, Department of Regenerative Medicine and Cell Therapy, Transplantation Biology Research Lab
G.R. Doshi and K.M. Mehta Institute of Kidney Diseases & Research Centre (IKDRC), Ahmedabad- 380016, Gujarat, India
Tel:
+91 79 22687387
Fax: +91 79 2268 5454
E-mail:
[email protected]

Received: March 21, 2017 Accepted: April 12, 2018 Published: April 17, 2018

Citation: Gupte KS, Vanikar AV, Patel CN, Bhargav AG, Patel JV, et al. (2018) Optimization of In vitro Culture Conditions of Interleukin-10 Secreting B Regulatory Cells of Human Origin. Cell Biol (Henderson, NV) 7:1. doi: 10.4172/2324-9293.1000139

Abstract

Abstract
Aim: 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 At each time point, one plate was withdrawn and contents centrifuged.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.

Keywords: B-regulatory cells; Lipopolysaccharide E. coli (LPS-EK); IL-10; Optimization; Characterization; Flow cytometry; Immunophenotyping; Adipose tissue derived mesenchymal stem cells (AD-MSC)

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