**4. Conclusions**

In this study, we presented a microfluidic chip with a nanofiber scaffold. Microfluidics can introduce dynamic conditions, and a nanofiber scaffold provides a 3D environment to an in-vitro cell culture. Moreover, the perfusion method in the chip allows the real-time monitoring of cell status with the conditioned culture medium. The microfluidic chip structure was fabricated using well-established soft-lithography at a preliminary level, and a nanofiber scaffold was prepared using electrospinning. In particular, the nanofiber scaffold had various diameter fibers. It was highly porous and heterogeneous in morphology, making it appropriate to provide a human body's ECM-like 3D environment. To justify the developed chip, HepG2 cells with various protein syntheses and 3D culture-specific morphology were cultured in the chip for 14 days [35]. Results demonstrated that the developed chip had excellent viability (higher than 95%) for HepG2 cells through live and dead cell assay. Additionally, the perfusion-based dynamic culture had a positive effect on the proliferation of HepG2 cells, which was about two-times higher than that under static culture condition. Finally, we presented the application of the chip to 3D cell culture and real-time monitoring based on conditioned culture medium. The cells cultured in the chip successfully formed engulfed aggregates (or large spheroid), which are a 3D culture-specific morphology of the cells. Moreover, albumin levels and AFP secretion from the cells were successfully monitored for 14 days. We expect the concept of the developed chip to be useful for studying 3D cell cultures, live assay or real-time monitoring of cell activity, and in-vitro drug screening and toxicity testing.

**Author Contributions:** Methodology, S.J.; validation, S.Y. and J.-Y.K.; formal analysis, J.H.K. and J.Y.P.; investigation, J.H.K.; writing—original draft preparation, J.H.K. and Y.H.J.; writing—review and editing, J.H.K. and Y.H.J.; supervision, Y.H.J.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2012-0009665, 2015R1A2A2A01005515, 2018R1A2B2009540).

**Conflicts of Interest:** The authors declare no conflict of interest.
