On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

Abstract

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.

Spatially and temporally controlled immune cell interactions using microscale tools

“…The first group of such systems utilize high-density hydrodynamic cell trap arrays within flowthrough channels [28][29][30][31]32 ] (Figure 1d). In particular, we developed a method, termed 'microfluidic cell pairing' [29,31,32 ], that uses microfabricated capture-cups to properly isolate and pair thousands of cells in parallel using a multistep cell loading procedure.…”

Droplet microfluidic platform for cell electrofusion

“…After counting on average 500 droplets per experiment (n = 5), of which 15% (75 droplets) contained two cells and qualified for possible electrofusion, 19 successful electrofusion events were found resulting in 5 % electrofusion efficiency. The established efficiency is comparable to the efficiency of both commercially available methods [53][54][55] as well as on-chip methods 52,[56][57][58] . To compete with existing methods, the electrofusion efficiency must be further improved, and there are several realistic opportunities for this.…”

“…The electrode width can also be adjusted, but should not be more than the droplet length, as the droplet must be in contact with both electrodes to create the electric field over the encapsulated cells. Additionally, previous results showed that applying multiple pulses (hence employing multiple electrode pairs) resulted in higher fusion efficiencies 52 .…”

“…β-dispersion arises when the numerous intracellular lipid cell membrane structures can be charged and discharged. The γ-dispersion is seen around 10 9 Hz and is caused by dipolar re-orientation of water 52 .…”

“…The conductivity is a measure for how strongly a given material allows the flow of electric current. In other words, a material has a high conductivity when the current density in response to an applied electrical field is high 52 .…”