*2.5. Functional Assays*

To verify hepatocyte viability of the developed microfluidic chip, the cells on the scaffold after settling down were stained with Calcein-AM to indicate live cells and ethidium homodimer-1 to indicate dead cells, according to the manufacturer's instructions (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA). The live and dead cells were observed using a confocal microscope (Carl Zeiss, Oberkochen, Germany). We investigated cell proliferation behavior under static and perfusion conditions to justify the effect of medium perfusion with microfluidic chip. Unlike cell culture under perfusion condition, the medium in the chamber was exchanged with fresh medium daily by using a pipet to compare cell proliferation under both conditions. Cell proliferation was assessed by measuring the DNA content of the cultured cells in each chip. The DNA content was measured on days 1 and 14. DNA from cultured cells was extracted using a commercial kit (Qiagen, Hilden, Germany). The DNA contents for both static and perfusion conditions were quantified by spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA).

Here, we quantified the secretion of albumin and alpha-fetoprotein (AFP) using enzyme-linked immunosorbent assay (ELISA) to demonstrate the applicability of the developed microfluidic chip to the 3D cell culture and real-time monitoring with HepG2 cells. Therefore, the conditioned culture medium was continuously collected at a rate of 0.1 mL/h and harvested daily by changing the syringe in the syringe pump. The amount of albumin secreted from the HepG2 cells was quantified every 2 days until day 14 using a human albumin ELISA kit (Bethyl Laboratories, Montgomery, TX, USA). AFP secretion was measured using an AFP ELISA kit (CUSABIO, Wuhan, China) every 3 days, from day 9 to day 14, according to the manufacturer's instructions. The required volume per sample of the conditioned medium for the assays was 100 μL, and the volume of the daily collected medium (2.4 mL) was sufficient to analyze various secreted proteins, up to 12 types, after duplication.

The morphology of the cells cultured in the microfluidic chip was investigated by SEM and fluorescent imaging. Cells cultured in the chips for 1 and 14 days were harvested with phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde solution. The samples for SEM were dried in a freeze-dryer (Labconco, Kansas City, MO, USA). Sample morphology was observed using SEM (Hitachi Hitachi, Tokyo, Japan). The cell morphology in the chip was assessed by staining F-actin with the phalloidin-fluorescein isothiocyanate (Phalloidin-FITC, Sigma-Aldrich, St. Louis, MO, USA) and nuclei with DAPI (4 ,6-diamidino-2-phenylindole, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA). The cells were then visualized using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
