HepG2 Cell Proliferation and Viability Assays

For all cell assays, HepG2 at passages below 10 were seeded in the gel at a final concentration of 1 × 10⁶ cells/ml and experiments carried out for n = 3. Proliferation of HepG2 in culture was assessed with a CellTiter Blue assay (Promega Corporation, Fitchburg, United States of America). 100 µl of medium with 20 µl CellTiter Blue were added to each well and incubated at 37°C for 3 h. Fluorescence intensity of the supernatant was read with an Infinite M Plex plate reader (Tecan Group AG, Männedorf, Switzerland). Cell viability was determined by staining for live cells with fluorescein diacetate (Sigma-Aldrich, St. Louis, United States of America) and dead cells with propidium iodide (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) (1:60 diluted in Ringer’s solution). Quantification of viable and dead cells was done in ImageJ (see Supplementary Figure S3).
For the analysis of live cell morphology inside the gels, HepG2 were incubated with 2 µM CellTracker™ Green CMFDA Dye (Thermo Fisher Scientific Inc. Waltham, United States of America) per 10 × 10⁶ cells for 30 min before seeding. Cells retained their fluorescence signal and passed it on to daughter cells for up to 7 days. Before immunofluorescence stains, cells were fixed in 4% paraformaldehyde (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 15 min and permeabilized with 0.5% Triton X-100 in PBS (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 10 min. Actin filaments were stained for 30 min with Alexa Fluor 488 Phalloidin (1:400 dilution in PBS) (Thermo Fisher Scientific Inc. Waltham, United States of America) and nuclei for 3 min with DAPI (1:800 dilution in PBS) (Sigma Aldrich, St. Louis, United States of America).