Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates

Abstract

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.

Keywords: cell culture; cryofixation; cryosections; electrospinning; freeze- substitution; freeze-fracture replica labelling; gelatin nanofibres; high-pressure freezing; immunogold.