*2.2. Fabrication of Microfluidic Chip Structure*

The microfluidic chip structure was made of PDMS using well-established soft lithography and micromolding [28,29] at a preliminary level. The master molds were prepared by cutting adhesive tape (3M) according to the channel shape, and removing the tape from all areas except the channel shape on a slide glass (ThermoFisher Scientific, Waltham, MA, USA)). PDMS (Dow Corning, Midland, MI, USA) was cast on the master molds using the pre-polymer (base) to curing agent weight ratio of 10:1, and the molds were cured in a dry oven at 60 ◦C for 3 h.

In the microfluidic layer, the microchannels had a cross-section of about 300 × 100 μm and a length of about 10 mm, whereas the chamber hole had a rectangular shape with a size of about 12 × 12 mm and a height of about 3 mm. The microchannels were patterned using soft lithography (Figure 2). After curing, a rectangular chamber hole was made by cutting a rectangle in the cured PDMS slab. A hole to the inlet microchannel was made by using a biopsy puncher.

**Figure 2.** Fabrication of patterned microchannels using soft lithography.

All the PDMS structures and slide glass were autoclaved for sterilization and bonded together after corona treatment (Electro-Technic Products Inc., Chicago, IL, USA) to secure tight bonding (Figure 1). Before inserting the nanofiber scaffold into the chamber, the scaffold was sterilized in 70% ethanol and thoroughly washed three times with PBS. A nanofiber scaffold was inserted into the chamber inside the bonded PDMS. The assembled chip samples were stored on a clean bench for a while. After seeding the cell suspension onto the nanofiber scaffold, the chamber was covered with glass coverslips. The microfluidic device was immobilized with two polycarbonate (PC) plates and screws to ensure no leakage.
