On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation.
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.
BIOS, Lab on a Chip group, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands. e.w.m.kemna@ewi.utwente.nl
Evelien W M
EW
Kemna
Floor
F
Wolbers
Istvan
I
Vermes
Albert
A
van den Berg
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology*; D002459:Cell Fusion / Q000295:instrumentation* / Q000379:methods; D002469:Cell Separation / Q000379:methods; D055664:Electrochemical Techniques / Q000295:instrumentation*; D006801:Humans; D006825:Hybridomas / Q000166:cytology* / Q000502:physiology; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D009101:Multiple Myeloma / Q000473:pathology*