Optimization and evaluation of an in vitro model of PEG-mediated fusion of nerve cell bodies

Abstract

Polyethylene glycol (PEG) is a cell membrane fusogen (de StGroth et al., 1980; Hoehn et al., 1978 [2]; Pontecorvo et al., 1975 [3]). In clinical application PEG allows some axonal fusion in peripheral nerve repairs, resulting in retention of some mobility and sensation during the regeneration process and reducing muscular atrophy. Several manuscripts exist concerning in vivo models of PEG fusion, while there is a distinct lack of in vitro studies. This study optimizes an experimental in vitro PEG fusion procedure in B35 rat neuroblastoma cells, through alteration of PEG and dimethyl sulfoxide (DMSO) concentrations and utilizes flow cytometry for assessment. The optimized procedure was then tested on B104-1-1 cells, a mouse neuroblastoma line expressing P2X7R, to validate our in vitro procedure/model against previous in vivo testing of P2X7R modulators, Brilliant Blue FCF (FCF) and bzATP. In brief, two cell populations were differentially stained, mixed, pre-treated (if applicable) and PEG-fused. Initial optimization generated the highest fusion rates with 70% PEG + 0% DMSO. Subsequent testing of the model with FCF and bzATP proved that assessment over time is required when evaluating potential modulators of PEG fusion. Our conclusion, at 72 h post PEG fusion, is that the optimized procedure is suitable for initial candidate testing of modulators of PEG fusion. In vitro experiments with FCF and bzATP corroborated previous in vivo findings and confirmed that DMSO is not required to aid PEG fusion in the in vitro model. The development of this cellular based in vitro model will be invaluable as a substitute for initial assessment of chemical modulators of PEG fusion.