*3.2. Application to 3D Cell Culture and Real-Time Monitoring*

The cell viability of a newly built culture system should be determined. Here, we investigated HepG2 cell viability of the microfluidic chip, embracing a nanofiber scaffold via live and dead cell assay. Figure 5 shows the assay results, in which the live cells were stained green, and the red ones corresponded to dead cells. The cell counting assay revealed that the developed microfluidic chip had sufficiently high viability for HepG2 cells (>95%).

**Figure 5.** Live and dead cell assay results of the developed microfluidic chip for HepG2 cells (day 1).

Figure 6 shows the comparison results of the proliferation of HepG2 cells cultured under static and perfused conditions for 1 and 14 days. As shown in Figure 6, the cells cultured for 14 days in the chip successfully proliferated regardless of the medium supply method used, although the standard deviations looked relatively large. The large standard deviation possibly resulted from the variation in cell seeding quality because of the innate uneven fibrous morphology. Even though the cells cultured under static condition revealed excellent proliferation for 14 days (6.6-fold), the perfused condition promoted cell proliferation more. The cells cultured under perfused condition proliferated by more than 12-fold during the same period. Thus, perfusion culture condition in the developed chip gave rise to about two times higher cell proliferation than the static culture condition (*p* < 0.001).

**Figure 6.** DNA content analysis of the cells cultured under static and perfusion conditions for 1 and 14 days in the chip.

The results demonstrated that the perfusion culture in the developed chip could provide cell-friendly conditions to the cells. To justify the applicability of the developed chip, 3D cell culture and real-time monitoring of the cells' status using the developed chip were carried out. Figure 7 shows the HepG2 cells cultured for 1 and 14 days in the chip with perfusion. As shown in Figure 7a,b, the cells with the cell length of 16.47 ± 4.84 μm successfully attached and hung on the nanofibers in the form of small spheroids, with diameters ranging from 20 to 50 μm on day 1. Moreover, the cytoskeleton organization, visualized by actin staining, revealed that actin filaments stretched along the nanofibers (Figure 7c). After 14 days, HepG2 cells re-established cell–cell contact and formed about 300 μm-diameter aggregates (or large spheroid) engulfed on nanofibers (Figure 7d–f), which has been reported to be the more desirable formation of HepG2 cells because of higher hepatocyte synthetic functions, higher response for drug toxicity, and better mimicking of in-vivo oxygen gradient in the hepatic lobule [3,24,33]. These results were far from that under 2D culture conditions based on a flat surface. In 2D conditions, HepG2 cells grow as monolayers with flat morphology, which results in short-lived canalicular-like structures [24]. Therefore, the developed microfluidic chip with nanofiber scaffold could successfully provide a 3D cell culture environment.

**Figure 7.** Confocal microscopy and SEM images of the cells cultured on the nanofiber chamber with microfluidic system for 1 (**a**,**b**,**e**) and 14 days (**c**,**d**,**f**). Especially, (**b**,**e**) are enlarged images of (**a**,**d**). The scale bars in the confocal microscopy images are 100 μm (**a**,**d**) and 20 μm (**b**,**e**).

The typical specific functionality of HepG2 cells was evaluated through real-time monitoring of the concentration of secreted albumin and AFP in the conditioned medium for 14 days. Albumin, which is secreted from HepG2 cells from human hepatocytes, is a marker of metabolic activity of hepatocytes in vitro, and it can indicate liver-specific functions [24]. Thus, the amount of albumin secreted from the cell cultured for 14 days in the chip was examined every two days (Figure 8a). The albumin production rate consistently increased from 3.07 ± 0.61 ng/mL at day 1 to 42.09 ± 1.91 ng/mL at day 14. Moreover, we monitored AFP secretion from the cells because AFP is known as a tumor-associated protein and important indicator of the differentiation of hepatoma cells and disease progression [34]. As shown in Figure 8b, the increase in secreted AFP level was similar to that in albumin secretion for the same duration. The AFP concentration in the conditioned medium collected at day 2 was 15.45 ± 5.13 ng/mL, and this value increased to 118.54 ± 3.19 ng/mL at day 14. As shown in these monitoring results, the developed microfluidic chip embracing nanofiber scaffold enabled the real-time monitoring of cell status based on the conditioned culture medium.

**Figure 8.** Real-time monitoring of secretion of (**a**) albumin and (**b**) AFP for 14 days.
