Minimum essential medium eagle

We used the human hepatoblastoma
HepG2 cell line (American Type Culture Collection, ATCC, Manassas,
VA, USA) and human hepatocellular carcinoma cell line Alexander (PLC/PRF/5,
ATCC, Manassas, VA, USA) in this study. Cell cultures were cultivated
in Minimum Essential Medium Eagle (BioConcept Ltd., Switzerland) supplemented
with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, US), 1% l-glutamine 100× (200 mM stock, Serana Europe GmbH, Germany),
and 1% penicillin/streptomycin (Thermo Fisher Scientific, US). Cell
cultures were kept in a humidified 5% CO₂ atmosphere at
37 °C. The culture medium (EMEM) was changed once per week. Cells
were regularly checked for common culture contamination such as mycoplasma
using the MycoAlert Detection Assay (Lonza, Switzerland). All cell
lines were authenticated by short tandem repeat (STR) DNA profiling
(ATCC, Manassas, VA, USA).
Approximately 10⁵ HepG2
and Alexander cells in a volume of 30 μL were seeded into porous
collagen scaffolds. CSs were placed in a 12-well plate. After 1 h,
the fresh medium was added to cover the whole surface of CSs. For
all utilized assays in the study, cells were cultivated in CSs for
7 days in a humidified 5% CO₂ incubator at 37 °C,
and the culture medium was changed every other day. Cells seeded on
a standard glass-bottom dish (Cellvis, Mountain View, CA, USA) with
a 35 mm diameter served as the monolayer culture (MC) model.

HeLa cells were obtained from the American Type Culture Collection (ATCC CCL-2). Cells were cultured in Minimum Essential Medium Eagle (BioConcept Ltd., Switzerland) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, US), 1% l-Glutamine 100 ×, 200 mM (Serana Europe GmbH, Germany) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific, US). Cell cultures were cultivated in a humidified 5% CO₂ atmosphere at 37 °C. Cell culture medium was replaced once a week. Cells were regularly checked for common culture contamination, such as Mycoplasma using MycoAlert Detection Assay (Lonza, Switzerland). HeLa cell line was authenticated by short tandem repeat (STR) DNA profiling (ATCC, Manassas, VA, USA).