Efficient and reliable establishment of lymphoblastoid cell lines by Epstein-Barr virus transformation from a limited amount of peripheral blood

Abstract

Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip^® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood.

Figure 1. Study design for the comparative experiments.

To evaluate the efficiency and reliability of the hemolytic protocol (flowchart, left side), a quantitative comparison was made with the gradient protocol (flowchart, right side) by changing the starting blood volume (5 ml, 2 ml, or 0.1 ml) with respect to the following three categories: (a) Comparison of the features of the cells (cell recovery, viability, and proportion of cell components) of the isolated WBCs prepared by the hemolytic protocol or PBMCs prepared by the gradient protocol, (b) Comparison of the growth curves of establishing LCLs (ellipse) from WBCs (LCL-hemolytic) or PBMCs (LCL-gradient), and (c) Comparison of the quality of genomic DNA (rectangle) based on the SNP genotype data (central arrow).

Figure 2. Growth curve of LCLs established from different blood volumes.

The LCLs were established by either hemolytic (LCL-hemolytic, solid black circle) or gradient (LCL-gradient, solid gray square) protocol from the peripheral blood of 5 ml (a), 2 ml (b), or 0.1 ml (c). Each LCL was cultured until 8 weeks and observed at 2-week intervals. Each point indicates the average and standard error bar of 10 LCLs. The vertical axis indicates the total number of viable cells and the horizontal axis indicates the weeks post EBV infection.

Figure 3. Genotype concordance by pairwise analysis.

Genotype concordance between the genomic DNA from LCL-hemolytic and the peripheral blood, from which the LCLs were established, is shown as box plots with median values and interquartile range of pairwise distances. These box plots were generated using 9 samples from sample groups #7, #8, and #9 (Table 1). When the LCL-hemolytic samples maintain the genomic DNA intact, the pairwise distance should be close to 0. The vertical and horizontal axes indicate pairwise distance and the filtering conditions, respectively.