C2 c2si

Cell survival in the 3DP-HepG2 model was evaluated immediately after printing to assess the effect of the manufacturing process, particularly the hydrogel composition and temperatures of the nozzle and forming space, on cell viability. A fluorescent live/dead assay was performed to determine cell survival. Briefly, a mixture of calcein-AM (1 μmol/L; Sigma) and PI (2 μmol/L; Sigma) was prepared and passed through a 0.22-μm filter prior to staining. The 3DP-HepG2 model was gently washed with phosphate buffered saline after crosslinking and immediately incubated in a calcein-AM/PI mixture for 15 min at 20–25°C in the dark. After incubation, the 3DP-HepG2 model was gently washed with phosphate buffered saline and observed under a laser scanning confocal microscope (C2/C2si; Nikon, Tokyo, Japan). Five random fields were captured for each sample, and cells in five samples were counted using ImageJ. Cell viability was calculated by counting the number of cells as follows: (live cells/total cells) × 100%.

After the respective treatments, lipid peroxidation was determined using the Bodipy C11 probe. To this aim, cultures were incubated for 30 min at 37 °C in the dark with 5 µM Bodipy C11. Then, cells were washed two times with PBS and were fixed for 15 min at 4 °C with 4% paraformaldehyde. After washing three times with PBS, the cells were labeled with the MAP2A antibody (to identify neurons), mounted on coverslips, and observed under the microscope. A confocal microscope (C2/C2si, NIKON) with a 40-oil immersion objective was used to acquire images by laser excitation at 405, 488, 561, and 640 nm. The ImageJ software was used for the generation of z-projections (sum of maximum intensity) from seven stacks (1 µm thickness each). Cells of interest were selected by delineation based on MAP2A staining; mean fluorescence intensity was measured for the green and red images, and the background was subtracted in both channels. Upon oxidation, the excitation maximum of the Bodipy C11 probe downshifts from 581 nm to 500 nm and the emission maximum from 591 nm to 510 nm. The oxidation ratio of C11 BODIPY 581/591 was calculated as an indicator of lipid peroxidation per cell following the equation:
In this equation, Bodipy^red corresponds to the non-oxidized fraction of the probe and Bodipy^ox corresponds to the oxidized fraction.

Cells were seeded on glass coverslips in a 12-well plate at a density of 5 × 10⁵ cells per well, and paraformaldehyde fixation was performed after treatment. Afterwards, these cells were immunostained overnight with primary antibodies against NFkB p65 (CST, USA). Then fluorescent secondary antibodies were co-incubated at room temperature for 30 min. After being rinsed, the cells were stained with DAPI (Sigma, USA) for 3 min and sealed. Finally, the cells were observed and imaged under a laser confocal microscope (Japan, Nikon, C2+/C2si+).