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.
Deskii sputter coater
Manufactured by Denton
The Denton DeskII Sputter Coater is a compact, versatile laboratory instrument designed for thin-film deposition. It utilizes a sputtering process to uniformly coat samples with a wide range of materials, including metals, alloys, and dielectrics. The DeskII features a user-friendly interface and automated controls for precise thickness control and repeatable results.
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2 protocols using deskii sputter coater
1
Confirmation of Scaffold-Free Cell Sheets
2
Gallbladder Wall Ultrastructure Analysis
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Additional sections of gallbladder wall were collected separately at necropsy and fixed in 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer at 4°C for 24 h then submitted to the Iowa State University Microscopy and NanoImaging Facility to be prepared for scanning electron microscopy (SEM). A single sample representative of each time point (2 h and 24 h) was selected for further processing based on evidence of normal mucosal architecture present in the histopathology examination. Fixed samples were rinsed in deionized water and post-fixed in 2% aqueous osmium tetroxide followed by dehydration in a graded ethanol series up to 100% ultra-pure ethanol and dried using a Denton DCP-2 critical point dryer (Denton Vacuum, Moorestown, NJ). When dried, the samples were placed onto adhesive coated aluminum stubs, sputter coated (Denton Desk II sputter coater, Denton Vacuum) with palladium/gold alloy, and imaged using a JEOL 5800 LV scanning electron microscope (Japan Electron Optics Laboratory, Peabody, MA) at 10 kV.
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