Isolation of B-1 subtype B lymphocytes from human cord blood

Abstract

Introduction: B-1 lymphocytes with high levels of CD5 markers are capable of differentiation and transformation into macrophage-like cells, as B-1 cells. This study aims to isolate and purify this unique subset from umbilical cord blood cell. Materials and Methods: 14 umbilical cord samples from healthy neonates of natural or elective cesarean deliveries were used. White blood cells were separated from red blood cells using hydroxyethyl starch or gelatin 3%. Cell culture was performed to separate adhesive and non-adhesive cells. Umbilical cord lymphocytes subsets were separated from the remaining leukocyte contents by Ficoll densitygradient centrifugation. Lymphocyte rosette test was used to assess purification stage. Results: Despite lack of difference in cell count between the two blood dilution methods, cell vitality in hydroxylethyl starch method was less than 87%, while cell vital activity in the dilution method by gelatin 3% was measured between 90 and 95%. Lymphocyte rosette test results indicate B-1 cell purification rate of approximately 99% by using the above method. Conclusion: This process indicates most of the extracted B-cells from human umbilical cord blood belong to the B-1 cell subtype, and gelatin 3% procedure is the best and most appropriate method in terms of number and vitality of cells.