2.7 Cell characterization
The viability of cells cultured on the SA/Gel and A-SA-Gel hydrogel samples was tested by Live/Dead staining assay (Biovision Inc., USA) according to the manufacturer’s recommendations, after 4 days of culture. The assay kit contains Calcein-AM, a cell-permeable green fluorescent dye (Ex = 490nm, Em = 515nm), to stain live cells, while the dead cells were stained by red fluorescent dye called propidium iodide (PI). Briefly, the staining assay was prepared using a mixture of 1 µL of Calcein-AM and 5µL of PI in 1 mL of staining buffer according to the manufacture’s protocol. The samples were removed from the culture medium after culturing 4 days and gently washed with PBS. Then, the prepared staining assay was added directly to the samples and incubated for 15 min at 37 °C. The stained samples were mounted on a microscope slide for imaging under an inverse fluorescence microscope (Eclipse Ti-U, Nikon Instruments Inc. Japan).
In addition, the HUVECs culture on the SA/Gel and A-SA-Gel hydrogel samples were stained with 4’,6-diamidino-2-phenylindole (DAPI) and tetramethylrhodamine (TRITC) phalloidin to visualize the cell nucleus and cytoskeleton, respectively. Briefly, the cell cultured samples were rinsed 3 times in PBS, fixed in situ with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min. Then, 100 μL of TRITC−phalloidin was gently added to the samples until it covers completely, and then it was incubated in a dark environment for 30 min at room temperature to stain the cell cytoskeleton. After that, the samples were washed 3 times in PBS, and 100μL of DAPI solution was added to the samples for 30 s to stain the cell nucleus. The fluorescence images were captured by a fluorescence microscope. Moreover, the cell adhesion morphology on the surface of the A-SA-Gel hydrogel samples was observed by using SEM after culturing for 4 days. Briefly, the cell-laden scaffolds were removed from the culture medium and gently washed with PBS, then immediately fixed with 4% paraformaldehyde for 1h, and washed with PBS three times. After that, the samples were immersed in different concentrations of ethanol (25%, 50%, 75%, 85%, 95%, and 100%) for 5 min for dehydration. The samples were then immersed in the mixed solutions of the absolute ethyl alcohol and hexamethyl disilylamine (3:1, 1:1, 1:3, and 0:1) for 10 min, followed by sputter coating with gold before imaging.
The quantitative analysis of the cell viability/proliferation was performed using Cell Counting Kit-8 (CCK-8) assay. CCK-8 allows sensitive colorimetric assay for the determination of the number of viable cells. Briefly, the SA/Gel and A-SA-Gel hydrogel samples were sterilized by 75% ethanol and added to a standard Petri dish (Corning, NY) with fresh culture medium for 2 h under the UV light. Then, the samples were added to the wells of 96-well plates (50 µL per well, Corning, USA). The prepared cells suspension was added into each well with printed samples at the density of 1×105 cells/mL. Three rows of the well of each plate were covered in every group. The cells were incubated at 37 °C in a humid atmosphere with 5% CO2. After 1, 3, and 5 days of incubation, 10μL of CCK-8 solution was injected into each well and incubated for 2 h in a CO2 incubator at 37 °C. The absorbance of 450 nm was determined by using a microplate reader (Infinite 200Pro, Tecan Group Ltd., Switzerland). The SA/Gel hydrogel sample was also tested as a control group. In addition, the cells suspension without a sample as a positive control group and only contain CCK-8 and medium as a background group.