Autosamdri 815b apparatus

To confirm densely adherent cells without an artificial scaffold in a cell-sheet and ECM deposited on the basal surface of a cell-sheet, cell-sheets were visualized with scanning electron microscopy (SEM) to confirm that: 1) cells were densely adherent despite lacking an artificial scaffold, and 2) ECM was deposited on the basal surface of the cell-sheet. For comparative purposes, “SMC only” cell-sheets were included in this experiment. Samples for SEM were fixed for 24 h at 4°C with 4% Paraformaldehyde and 2% Glutaraldehyde in 0.1 mol/L Sodium Cacodylate Buffer (pH 7.2) after lifting up a bi-level cell-sheet from the Upcell dish. Cell-sheets were rinsed in the same buffer and post-fixed for 1 h with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50, 70, 90, 100% [2x]; 10 min each), samples were critical point dried with liquid CO₂ in a Tousimis Autosamdri-815B apparatus (Tousimis), mounted with colloidal graphite on 15-mm aluminum stubs (Electron Microscopy Sciences) and sputter-coated with 50 Å of gold-palladium using a Denton DeskII Sputter Coater (Denton Vacuum). Visualization was performed with a Zeiss Sigma FESEM (Carl Zeiss Microscopy) operated at 2–3 kV, using inLens SE detection at a working distance of 5–6 mm. Images were captured in TIFF format using a store resolution of 2048 × 1536 and a line averaging, noise reduction algorithm.

For SEM analysis, samples were fixed for 24hrs with 4% paraformaldehyde and 2% glutaraldehyde in 0.1M Sodium Cacodylate Buffer (pH7.2), rinsed in the same buffer and post-fixed for 1hr with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50, 70, 90, 100 %(twice); 10 min each) samples were critical point dried with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis, Rockville, MD), mounted onto double-sided copper tape on 15mm aluminum stubs (Electron Microscopy Sciences, Hatfield, PA), and sputter-coated with 50Å of gold-palladium using a Denton Desk II Sputter Coater (Denton Vacuum, Moorestown, NJ). For cross-sectional views samples were cut with a single-edge razor blade, and mounted onto low profile 45° / 90° SEM stubs (Ted Pella, Redding, CA) before sputter-coating. Visualization was performed with a Zeiss Sigma FESEM (Carl Zeiss Microscopy, Thornwood, NY) operated at 2-3kV, using InLens SE detection at working distance 4-5mm. Images were captured in TIFF using store resolution 2048×1536 and a line averaging noise reduction algorithm.