Hybridoma Production

 Electrofusion (Electro Cell Fusion)

Electrofusion was performed using the ECFG21 or the LF201 Electro Cell Fusion Generator (Nepa Gene, Japan) and the CUY497P2 MS Stand Model Chamber Type Platinum Electrode, L80mm x W2mm x H5mm, 0.8ml (Nepa Gene, Japan), (Fig. 1). The electrofusion buffer was composed of 0.3M mannitol, 0.1mM calcium chloride and 0.1mM magnesium chloride.

1) Using a ratio of one-to-one, B lymphocytes from animals immunized with ovalbumin and myeloma cells were put into a 15ml centrifuge tube containing electrofusion buffer and centrifuged. The supernatant was removed and the cells were suspended with 2.4ml of electrofusion buffer solution.

2) Set the Generator as follows: AC (1MHz, 30Vrms, 20sec), DC (350V, pulse length: 30sec, pulse interval: 0.5sec, 3 times). Connect the CUY497P2 electrode.
Inject 0.8ml of the suspended cells into the CUY497P2 electrode. Press the start button and the AC current for 20 seconds will bring the cells into alignment (pearl chain), then the Generator will automatically apply DC pulses three times. Then remove cells from Electrode. Repeat the step 2 two more times.

3) Centrifuge for 5 minutes at 1,000 rpm. Mix cells with HAT medium add BM-Condimed H1 and inoculate into four 96 well plates.

 Comparison of methods using PEG and electrofusion

Experiments using PEG and electrofusion were done with iliac lymph node lymphocytes from mice. The cells in two cryogenic vials were thawed and mixed (approx. 4 x 107 cells). Then half of the cells were fused by PEG and the other half of the cells were fused by electrofusion. This set of experiments was done three times. The result was that the number of positive wells by electrofusion was 1.5-4.0 times as many as by PEG (Table 1). The average rate was 2.8 times.

Table1: Comparison of fusion methods using mouse iliac lymph node lymphocytes

 Comparison of methods using PEG and electrofusion

Experiments using PEG and electrofusion were done with iliac lymph node lymphocytes from mice. The cells in two cryogenic vials were thawed and mixed (approx. 4 x 107 cells). Then half of the cells were fused by PEG and the other half of the cells were fused by electrofusion. This set of experiments was done three times. The result was that the number of positive wells by electrofusion was 1.5-4.0 times as many as by PEG (Table 1). The average rate was 2.8 times.

 Discussion of electrofusion

Cell fusion with PEG, electrofusion and HVJ-E was done using iliac lymph node lymphocytes from rats (Fig. 2 and 3). Cell fusion by electrofusion can be done with smaller number of lymphocytes than by PEG and HVJ-E, and its operation time is shorter than PEG and HVJ-E. The growth of fused cells after electrofusion was faster because its damage seemed to be low. Therefore ELISA screening for positive wells by electrofusion was done one day earlier than by PEG. The fusion technique does not vary from individual to individual because electrofusion operation is simple. Once the fusion operation and electric setting are optimized, the fusion with the equal condition can be always generated for the same animal’s lymphocytes. The fusion with an equal condition leads to small variation in the data. With electrofusion, the fusion time is short, the cell damage is low and the fusion efficiency is the highest. At this experiment, the number of positive wells was 6 times better compared to PEG.

◆ Summary