The effect of support cells on B lymphocyte viability in an in vitro human immune system construct
Human B lymphocytes are notoriously difficult to culture. Two to three days after plating, a sharp decline in viability and cell number can be observed. The objective of this study was to evaluate the effect of support cells on B cell viability in an in vitro human immune system construct. B cells were combined with dendritic cells (DCs) and cultured for various periods of time in the presence of one of three types of support cells: EA cells, HS-5 cells, and HS-27 A cells. The B cells were either in physical contact with the support cells, or allowed to interact through soluble factors in the media in order to determine if the effect on viability was contact dependent or independent. Viability was assessed using flow cytometry.
Finally, two functional assays were performed to evaluate the ability of the cultured B cells to respond to an immune challenge. Both recall and nai've antigens were used. The B lymphocytes were then assessed for viability, proliferation and activation using flow cytometry. ELISPOT was also employed to determine if any antigen specific antibodies were produced by the B cells.
It was found that while the support cells did improve viability, they did not produce consistent or reliable results. Additionally, B lymphocytes cultured in the presence of support cells or support cell conditioned media had no antigen specific tetanus response and reduced proliferation. Therefore, even though the support cells did under some conditions enhance lymphocyte viability, the lack of a positive functional response negates the value of using them in an experimental system.
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Bachelor of Science (B.S.)
Burnett School of Biomedical Sciences
Molecular Biology and Microbiology
Biomedical Sciences -- Dissertations, Academic; Dissertations, Academic -- Biomedical Sciences
Length of Campus-only Access
Honors in the Major Thesis
Feldman, Kristyn, "The effect of support cells on B lymphocyte viability in an in vitro human immune system construct" (2007). HIM 1990-2015. 634.