*2.1. Concept of Microfluidic Chip with Nanofiber Mat*

Figure 1 shows the schematic concept of the proposed microfluidic chip embracing nanofibers, which consists of a nanofiber scaffold, a microfluidic chip structure, perfusion environment, and cells. The chip was designed to mimic the dynamic microenvironment of the human body, support perfusion-based long-term culture, and allow real-time monitoring of secretions and functionalities of the cultured cells in vitro. An electrospun nanofiber scaffold was introduced to a microfluidic chip to provide a 3D extracellular matrix (ECM)-like environment, because the nanofibers have similar morphology to the human body's ECM [25]. The nanofiber scaffold was located on the chamber bottom. The microfluidic chip has a simple structure composed of micro-channels, a chamber, and gate holes, and it is made of Polydimethylsiloxane (PDMS). The cell suspension was introduced into the top opening of the chamber before closing the opening with a cover slip. Fresh culture medium was supplied to the cell-seeded nanofiber scaffold via the inlet microchannel to provide supplied oxygen and nutrients to the cells. The cell culture-conditioned medium through the outlet microchannel was collected to monitor cell activity. Fluidic connections to the microfluidic chip were

made with tubing inserted through the inlet and outlet holes. Culture medium perfusion/flow in the chip was established with the help of a perfusion environment consisting of a fresh media reservoir at the side of the inlet and a syringe pump at the side of the outlet. Therefore, medium flow was derived by negative pressure. The collected conditioned medium in a syringe was easily transferred for analysis, such as enzyme-linked immunosorbent assay (ELISA).

**Figure 1.** Concept of a microfluidic chip with a nanofiber scaffold.

As shown in Figure 1, the chip is composed of four layers: a supportive plate (slide glass), two microfluidics layers (PDMS), and a window to chamber. The microfluidic layer has a microchannel on the bottom plane and a rectangular hole as a chamber at the center of the layer. A thin coverslip was used as the window to chamber in this study. After assembling all the layers, the rectangular holes formed the center chamber with the supportive plate and window to chamber.

Flow rate of the culture medium through the chip was determined by considering the required amount of culture medium for cell growth, 2D culture condition, and allowable shear stress on the cells induced by culture medium flow in microfluidics. Information on the required amount of culture medium can be obtained from the culture product company, whereas the shear stress limit can be obtained from a previous study [26]. The amount of media supplement per hour (flow rate) can be determined from the following equation:

$$\frac{M}{N\_R \times T} \times N\_{chip} \le \|q\| \le \frac{\pi\_{\text{max}} b h^2}{6\mu}$$

where *M* is the amount (volume) of culture medium supply for the number (*MR*) of cells at a given culture area. *T* is the medium exchange period. *M*, *NR*, and *T* are the recommended values under 2D culture condition. *Nchip* is the number of cells seeded in the chip, *τmax* is the allowable shear stress loaded to cells to avoid cellular damage, *μ* is the viscosity of medium (g/cm·s), *q* is the flow rate through the chip (cm3/s), and *b* and *h* are the width (cm) and height (cm) of the microchannel, respectively. The maximum limit of the flow rate, which was calculated from the allowable shear stress, was 1.54 mL/h. The minimum flow rate, which was calculated from the 2D culture protocol [27] considering the number of seeded cells in a scaffold and scaffold area, was 0.001 mL/h. We set the flow rate at 0.1 mL/h between the maximum and minimum limits.
