3.2. Immunostaining and live/dead staining assay
Live/Dead Staining Assay: The HUVECs were cultured in the RPMI 1640 medium supplemented with 10 v/v% FBS. The cells were resuspended in bioink to achieve a final cell density of 1 × 106cells/mL. Gel filaments of the cell hydrogel suspension were printed into 12-well plates at different pressures (20–40 kpa) by using a nozzle of diameter 1 mm. The cell viability was assessed after printing applying vital-fluorescence-staining using an inverted microscope (DMI6000B; Leica Microsystems, Wetzlar, Germany). The staining solution contained 10 µg mL−1 propidium iodide (Solarbio Inc.) and 10 µg mL−1 acridine orange (Solarbio Inc.). The number of living and dead cells was counted using the Image J software. Cell viability, (i.e. the ratio between living and dead cells) was plotted against the shear stress, which was calculated for each set of the printing parameters by using the fluid dynamics model.
Immunostaining: The samples were obtained 1, 2, and 3 days after printing. The samples were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, after which all the samples were rinsed with PBS. The cells were permeated with PBS with 0.1% Triton X (PBS-Tx-0.1%) and sealed in PBS containing 2.5% bovine serum albumin for 30 min to reduce nonspecific background staining. The cells were incubated for 2 h with F-actin (1:500, FITC phalloidin, Sigma, P1951). The cell nuclei were finally labelled using 4-6-Diamidino-2-phenylindol (DAPI, Thermo Fisher Scientific). The cells were rinsed twice and then incubated with DAPI for 10 min under room temperature. After the reaction, the fluorescence microscope was employed to image all the samples.
CCK-8: HUVECs proliferation was assessed using the cell counting kit-8 (CCK-8) method. The CCK-8 was purchased from Solarbio Inc. After the cells are cultured for a certain period (24, 48, and 72 h), they are washed with PBS solution and placed in a new 24-well plate. Then, the CCK-8 reagent was diluted in RPMI 1640 medium at the ratio of 1:10 by adding 200 μL of the diluent to each well and then incubating for 2 h. Next, 100 μL of the diluent from each well was taken and added to a 96-well plate. The absorbance was measured at 450 nm by using a microplate reader (ELX800; BioTek, Vermont, USA).