LIFE09200604007 Tabish - Homi Bhabha National Institute

Results
4.1.2 Establishment of LCLs and morphological analysis
A total of 34 LCLs from UADT MPN patients (n=24) and healthy controls
(n=10) were prepared from PBLs isolated from blood samples. Sixteen UADT MPN
patient (n=8) and healthy control cell lines (n=8) were prepared as a part of earlier
study in the lab (marked with * in Table 5 and Table 6, prepared by Ashwin Kotnis);
eighteen MPN patient (n=16) and control cell lines (n=2) have been prepared in the
present study. Blood sample (3 ml) from MPN patients and cancer free control
individuals was obtained and subjected to PBL isolation by ficoll-hypaque density
gradient centrifugation. Approximately 4x10 6 cells were obtained from 3 ml blood.
After lymphocytes isolation, LCLs were generated by infecting isolated
lymphocytes with EBV which is known to infect only B cells in a mixed population of
B, T and natural killer cells present in PBLs. Presence of complement receptor type 2,
commonly known as CR2 (CD21) on B cells creates a route for virus entry into the cell.
Considerable cell death of the PBLs was observed 24 h post EBV infection; however
virus infection promoted B cells to re-populate the culture. The time taken for each
LCL preparation varied, on an average culturing the cells 3-4 weeks post infection was
sufficient to produce >1 million cells. Once the cell lines started growing, initial
passage cells were cryopreserved for future use.
The LCLs in the present study were established as a model to study
carcinogen sensitivity in vitro and to understand the aetiology of UADT MPN. Hence it
was necessary to ensure that the process of EBV transformation did not affect the basic
genotypic and phenotypic properties of the cells and LCLs were comparable to isolated
lymphocytes. Therefore, random LCLs were undertaken for partial characterization
with respect to cell population doubling time (PD), ploidy status, cell surface marker
and expression and activity of ATM gene.
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