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. In vitro grown vertebrate cells are an indispensable tool for cell biological research, and are widely used for the subcellular localisation of macromolecules at the level of electron microscopy (EM). Yet, the fast and efficient harvest of living, adherent cells without affecting cellular morphology and physiology is still a not satisfactorily solved problem. Thus, the potential of recent improvements (1-3) of cryo-immuno-EM (cryo-IEM) has so far not been fully exploited.To begin with, immunogold-labelling of cryosections according to Tokuyasu (4) has been successfully combined with rapid cryofixation (2,3). The new 'hybrid' approach avoids artefacts resulting from conventional chemical fixation. It is based on cryofixation of native, unfixed specimens, followed by chemical stabilisation of cellular ultrastructure and antigenicity at around −90• C by means of freeze-substitution (FS) and, finally, sample rehydration and postfixation prior to cryosectioning (2,3). So far, this modified Tokuyasu-technique was mainly used for tissues and suspension cultures (2,3,5), but not regularly for adherent cell cultures, with very few exceptions, when relatively large, voluminous cells (HepG2) were cultured on gelatine beads (Cytodex™, Sigma: Ø ∼100 μm; refs. (2,6)). Yet, in our previous studies we found that commercially available beads are less suitable for applying this advanced IEMtechnique to flat and/or small cells, such as mouse embryonic fibroblasts (MEF) with a maximal height of ∼4 μm (ref.(7) and our unpublished data). Considering the adverse ratio of bead-diameter versus cell-height it is evident that most of the area of an average 400 × 400 μm-cryosection is occupied by section profiles of the carrier beads, but not by the cells under investigation. The analysis of a fair amount of cells, however, is mandatory for unbiased stereology (8). Furthermore, sampling of cells grown on beads prior to cryofixation requires time-consuming intermediate enrichment-steps, possibly leading to unwanted physiological and ultrastructural alterations.The second cutting-edge IEM-technique to mention here is sodium dodecyl sulphate-digested freeze-fracture replica labelling (SDS-FRL (1)). So far, SDS-FRL was predominantly applied to chemically fixed tissues e.g. (9); see also (10) for review. The few SDS-FRL studies on natively cryofixed monolayers relayed on...

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

“…Technical note : Cryo‐section‐IEM from cryo‐fixed, rehydrated samples worked here generally well for endo/lysosomes. However, it failed so far for certain small vesicular/tubular compartments around MVB 2 (which were, in addition, not always sufficiently contrasted, thus, not well visible and not recognized at all in chemically fixed samples).…”

“…MEF (passages 16–24) were grown under standard conditions in Dulbecco's modified Eagle medium (DMEM: #E15‐843, PAA‐Laboratories; or #D5546, Sigma‐Aldrich) supplemented with 10% (v/v) fetal calf serum (#A15‐151, PAA), 100 IU/mL penicillin (#15140‐130, PAA,) and 100 µg/mL streptomycin (#15140‐130, PAA). For EM, MEF were plated at days 10–12 after thawing onto sapphire coverslips or gelatin fiber matrices and cultured for 2–3 days until ∼70–80% confluence. This guaranteed optimal vitality of the cells, thus, reproducible results.…”

“…For immuno‐EM or cytochemistry, the FS medium comprised acetone plus 0.5% (v/v) glutaraldehyde, 0–0.05% (w/v) OsO 4 , 0.1% (w/v) uranyl acetate, 0.5% (v/v) methanol and 4% (v/v) water. In this case, freeze‐substituted samples were rehydrated and subjected to (i) thawed cryo‐section immuno‐labeling , or (ii) diaminobenzidine (DAB) cytochemistry (Ref. , modified after Ref.…”

“…In this case, freeze‐substituted samples were rehydrated and subjected to (i) thawed cryo‐section immuno‐labeling , or (ii) diaminobenzidine (DAB) cytochemistry (Ref. , modified after Ref. ), or (iii) silver enhancement for visualizing EGF‐Nanogold.…”

“…Indirect immuno‐gold labeling of thawed cryo‐sections from cryo‐fixed or chemically fixed samples was performed as described previously . Bound antibodies (see Table ) were visualized with secondary antibodies conjugated to colloidal gold (from British Biocell) or Ultra Small Gold™ that was subsequently silver enhanced with AURION R‐GENT SE‐EM silver (#500.033, AURION).…”