Journal of Isfahan Medical School

Journal of Isfahan Medical School

Isolation of Peripheral Blood Plasma Cells and Co-Culture with Bone Marrow Stromal Cells in Order to Long-Term Culture

Document Type : Original Article (s)

Authors
Masoumeh Bazzaz 1
Jalil Fallah-Mehrabadi 2
Mahdi Mahdavi 3
Mahdi Zeinoddini 2
1 Biosciences and Biotechnology Research Center, Malek Ashtar University of Technology, Tehran, Iran
2 Assistant Professor, Biosciences and Biotechnology Research Center, Malek Ashtar University of Technology, Tehran, Iran
3 Assistant Professor, Department of Immunology, Pasteur Institute of Iran, Tehran, Iran
Abstract
Background: Single B cell antibody technology is a novel strategy to make human monoclonal antibody (mAb). Its main part is long-term culture of primary plasma cells (PC) to secrete antibody. PC survival in bone marrow is related to environmental signals, which should supply using a feeder cell monolayer during in-vitro culture of PCs. It seems that among different kinds of feeder cells, bone marrow stromal cells (BMSCs) are the best. The aim of this study was isolation of antibody secreting plasma cells from peripheral blood and surviving them during in-vitro culture.Methods: After tetanus vaccination, Ig concentrations of serum samples were titrated using enzyme-linked immunosorbent assay (ELISA) method. Isolated peripheral blood mononuclear cells (PBMCs) of vaccinated donor were stained with three fluorescent anti CD38, anti CD19 and anti CD45 antibodies. Stained plasma cells were sorted into 96 wells using fluorescence-activated cell sorting (FACS) and cultured with BMSCs as feeder cell. After 10 days, in order to determine plasma cell survival, total RNA were extracted from plasma cells and reverse transcription polymerase chain reaction (RT-PCR) method was performed using antibody specific primers.Findings: 7 days after vaccination, the serum antibody titration of donor was 16 IU/ml, which confirmed provoking a good immune response against tetanus vaccination. Flow analysis of peripheral blood sample on seventh day showed the presence of 0.3% plasma cells in PBMCs. Using RT-PCR with primers specific for antibody gene, the amplification of a variable heavy chain (VH) gene segment with the size of 400 bp was done; this confirmed the plasma cells survival and antibody gene expression during co-culture with BMSCs.Conclusion: The main result of this project was finding suitable pattern to isolate and survive plasma cells during 10 days in-vitro culture. Therefore, a part of single B cell antibody technology was established. Some of the results consist of percentage of plasma cells in PBMCs and the size of VH segment are in agreement with previous studies.
Keywords
Human monoclonal antibody
Plasma cell
Bone marrow stromal cell
Fluorescence-activated cell sorting (FACS)
Co-culture
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Journal of Isfahan Medical School
Volume 32, Issue 282 - Serial Number 282
March and April 2014
Pages 534-543

  • Receive Date 09 January 2014
  • Accept Date 06 April 2022