Microstructure characterization with scanning electron microscopy (SEM) was carried out by adapting the protocols in [35 (link),41 (link)]. Briefly, COL1, HC-L-ECM and LC-L-ECM thin structures were fixed for 48 h in 4% PFA in PBS and then washed three times in 0.1 M phosphate buffer (PB) for 10 min. Resulting samples were incubated in 4% osmium tetroxide for 90 min followed by successive washes in deionized water until there was absence of osmium tetroxide. Then, samples were dehydrated in increasing concentrations of ethanol solutions and preserved in absolute ethanol at 4 °C and critical point dried using an autosamdri-815 critical point dryer (Tousimis, Rockville, MD, USA). Samples were mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA) for imaging and were carbon coated before imaging with a JSM-6510 (JEOL, Tokyo, Japan) scanning electron microscope at 15 kV. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to process the SEM images. Density of fibers per area was obtained by applying a threshold to isolate the fibers in a specific plane to quantify them, while diameter of fibers was estimated as the average of ten randomly-selected fibers in three different zones of each sample.
Conductive adhesive tabs
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Conductive adhesive tabs are a type of laboratory equipment that provide a conductive surface for various applications. They are made of a conductive material and are designed to adhere to a variety of surfaces. These tabs can be used to create electrical connections or to enhance the conductivity of a specific area.
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4 protocols using conductive adhesive tabs
1
Microstructural Analysis of Collagen Matrices
2
3D Bioprinted cECM Hydrogel Scaffold Ultrastructure
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The ultrastructure of the cECM hydrogel scaffolds was visualized with a JSM-6510 (JEOL, Tokyo, Japan) scanning electron microscope (SEM). cECM scaffolds of 20 × 9 × 3 mm3 were produced via 3D bioprinting casting
The 3D scaffolds were fixed in 4% paraformaldehyde (PFA) in PBS for 48 h and then washed three times with 0.1 M phosphate buffer (PB). Next, the samples were incubated in 4% osmium tetroxide for 90 min and then rinsed with deionized water. Subsequently, samples were dehydrated by washing them with ethanol 80% (×2), 90% (×3), 96% (×3), and 100% (×3) and preserved in absolute ethanol at 4 °C until critical point drying (Autosamdri-815 critical point dryer, Tousimis, Rockville, MD, USA). Samples were then carbon coated and mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA). Imaging was performed by using an SEM (JSM-6510, JEOL, Tokyo, Japan) at 15 kV.
The diameter of the fibers was assessed following the method developed in [47 (link)]. Briefly, 10 fibers of three different zones of each sample were randomly selected, and their diameter was computed with ImageJ Software v1.53 (National Institute of Health, Bethesda, MD, USA).
The 3D scaffolds were fixed in 4% paraformaldehyde (PFA) in PBS for 48 h and then washed three times with 0.1 M phosphate buffer (PB). Next, the samples were incubated in 4% osmium tetroxide for 90 min and then rinsed with deionized water. Subsequently, samples were dehydrated by washing them with ethanol 80% (×2), 90% (×3), 96% (×3), and 100% (×3) and preserved in absolute ethanol at 4 °C until critical point drying (Autosamdri-815 critical point dryer, Tousimis, Rockville, MD, USA). Samples were then carbon coated and mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA). Imaging was performed by using an SEM (JSM-6510, JEOL, Tokyo, Japan) at 15 kV.
The diameter of the fibers was assessed following the method developed in [47 (link)]. Briefly, 10 fibers of three different zones of each sample were randomly selected, and their diameter was computed with ImageJ Software v1.53 (National Institute of Health, Bethesda, MD, USA).
3
Structural Analysis of ECM Hydrogels
4
Hydrogel Preparation and Imaging
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The hydrogel was freshly prepared and fixed in glutaraldehyde 2.5% in phosphate buffer (PB) 0.1 M, pH 7.4 for 24 h. After the fixation, it was washed ×4 with PB 0.1 M and treated with osmium tetraoxide 1% in PB at 4 °C for 90 min. Then, the sample was cleaned with ultrapure water until no yellow color was observed in the sample and water. The sample was cleaned with ethanol 50% followed by ethanol 70% and kept at 4 °C overnight. Then, it was dried by using serial dilutions of ethanol from 80% to 100%. Finally, the sample was critical point dried (Autosamdri-815 critical point dryer, Tousimis, Rockville, MD, USA), gold coated, and mounted using conductive adhesive tabs (TED PELLA, Redding, CA, USA). Imaging was performed by using scanning electron microscopy (SEM) (JSM-6510, JEOL, Tokyo, Japan) at 15 kV.
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