s The GFP Viability Assay for Cryobiology Applications: A Mechanistic Understanding

The authors have previously implemented Green Fluorescent Protein (GFP) transfection as a marker to assess viability both in vitro and in vivo following freezing injury, with loss of GFP fluorescence following treatment indicating cell death. Although excellent correlations with conventional vital dyes and staining methods (membrane integrity, histology) were observed following injurious freezing, until now the basis for the loss of GFP fluorescence was not comprehensively explored. In this work it was hypothesized that membrane breach caused by freezing causes leakage of GFP. Diffusion of GFP into the extra-cellular space then causes a loss of intracellular and average fluorescent signal as the GFP is diluted and its fluorescent signal attenuated (diffusion-dilution hypothesis). A simple one-dimensional (1-D) mass diffusion equation implementing literature values of GFP diffusivity was found to adequately account for the observed time scale of GFP fluorescence attenuation in vitro. Conservation of mass was established by monitoring extracellular solution fluorescence before and after cell lysis, which is consistent with the hypothesis of simple diffusion of a stable GFP molecule from the intracellular to extracellular space. The effect of freezing on the protein, external to the cellular environment, was investigated by repeated freezing of aqueous solutions of purified recombinant protein. A significant difference (p < 0.01) in fluorescence intensity between control samples and the frozen protein solutions was not observed until the third freezethaw cycle. These results suggest that cold denaturation of the protein is not a major contributor to GFP fluorescence loss following lethal freezing of cells and that the diffusion and dilution of the fluorophore is the basis of fluorescence loss. The intracellular GFP thus functions as a membrane integrity indicator following low temperature freezing injury.