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

Abstract

Synopsis: Spiderweb-like gelatine matrices generated by electrospinning are introduced as versatile substrate for culturing animal cell monolayers destined for cryofixation and EM. Thus, non-disruptive sampling and transfer of native cells into high-pressure freezing devices is possible within about 30 seconds. In addition to cryosection- and replica-labelling, this aproach can be applied to various complementary microscopy and biochemical methods. 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. © 2013 John Wiley & Sons A/S.