Determination of cell fusion efficiency
Cells stained with red and blue dyes were used to determine the efficiency of cell fusion. The blue- and red-stained cells were used as fluorescence minus one (FMO) controls for fluorochromes PE and BD Horizon V-450 in flow cytometry, respectively. Eliminating the background noise of possible unstained cells, the mean percentage of the fused cells emitting both red and blue fluorescence simultaneously was 18.5% 4.60% (n = 3). Considering that self-fusions among dye-stained cells are also likely to occur and could not be detected using a flow cytometer, the suggested fusion efficiency was at least 13.9%.
Determination of ploidy
In a separate experiment, five single clones obtained from the PEG-treated cell population by ClonePix2, were randomly selected to determine the DNA ploidy using flow cytometry. From these data, it is evident that clone #1 was haploid compared to the diploid control (untreated) cells. While clones #2~#5 showed no significant difference in fluorescence intensity compared to the control group, the G2/M peaks of clones #2 and #3 appeared to be higher than that of control group. Given the fact that the DNA content of diploid G2/M is indistinguishable from that of tetraploid G0/G1 (DNA tetraploid), we proceeded to analyze metaphase cells of each clone to gain further insight into the chromosomes of the PEG-treated clones.
Analysis of chromosome number
In the control group, approximately 60% possessed 26-30 chromosomes, two out of five clones contained 19-20 chromosomes, whereas the rest contained 35-36 chromosomes (n = 50). This shows that while the control group displayed aneuploidy to a certain extent, each clone maintained a certain number of chromosomes regardless of the number of passages and culture time. On the other hand, the PEG-treated clones did not exhibit any consistency in chromosomal number and a wide range of aneuploidy was observed (Figure 1). Notably, three treated clones (#1, #2 and #3), showed ambivalent chromosome numbers, i.e. cells with less chromosomes (<16) coexisted with cells having higher number of chromosomes (>41) (red arrows in Figure 1). These characteristics were not observed in the control group. Hence, we deduced that PEG-mediated cell fusion induced chromosomal instability that is different from the one occurring naturally.
Evaluation of antibody concentration
The five cell lines obtained from PEG-treated group displayed distinct antibody-producing ability. Particularly, clones #1, #2 and #3, which exhibited ambivalent chromosome numbers, presented 1.82, 1.42 and 1.36 times higher final antibody concentration than the control cell pool, respectively. Specific antibody production rates of all these three clones were at least 1.57 times higher than that of control cell pool. Based on the fact that clones which exhibit ambivalent chromosome numbers showed higher final antibody concentration and production rate than the controls and PEG-treated clones #4 and #5, we suggest that diversity in chromosome number, a type of chromosomal instability induced by cell fusion is an essential factor in constructing high antibody-producing cell lines.