## 2 Fabrication of the PDMS CD-Adapter  
We fabricated an adapter with a small recess structure to control the flow of liquid in a culture dish in a closed environment. To fabricate a PDMS adapter that can be attached to a commercially available culture dish without gaps, a method using a culture dish equipped with a negative structure of a small recess structure as a mold was adopted. A U-shaped small recess structure (U-shaped structure) and an I-shaped small recess structure (I-shaped structure) as shown in Figure 1 were provided in a CD-adapter corresponding to a 35-mm culture dish. A semicircular arc with outer and inner diameters of 32 mm and 30 mm, respectively, and a rectangle with a width and length of 2 mm and 34 mm, respectively, were cut out from a 500-\(\upmu\)m thick polyethylene sheet (HRHG711303, KYOWA). The circular arc was pasted on the bottom of a 35-mm culture dish with a 2-mm gap from the edge, and the rectangle was stuck on a line bisecting the culture dish (Figure 2A). The outer periphery of the 35-mm culture dish with a mold structure was filled with epoxy resin (STYCAST 126) PTA, Henkel) for the bearing surface of the adapter. Therefore, as shown in Figure 2A, a 60-mm culture dish was used as a tray, being attached centered under the 35-mm culture dish. Epoxy resin was poured into the gap between both culture dishes, which were left for 9 h to stand at 25 \({}^{\circ}\)C to cure the epoxy resin (Figure 2B). After checking that the epoxy resin had cured, 2 mL of PDMS (CAT-106F, Shin-Etsu Chemical) was injected into the 35-mm culture dish and left to stand at 25 \({}^{\circ}\)C for 6 h to cure the PDMS (Figure 2C). The reason for setting the volume of PDMS to 2 mL is to make the thickness of the bottom membrane of the CD-adapter 2 mm. Because the bottom area of the 35-mm culture dish is 9 cm\({}^{2}\), the volume of PDMS required to make the bottom membrane thickness 2 mm is 1.8 mL. In this study, we set it to 2 mL for easy preparation of the experiments. After confirming that the PDMS had cured, the 35-mm culture dish and the center of a cylinder (aluminum cylinder, [PERSON], Tokyo, Japan) with a diameter of 25 mm was placed on the PDMS membrane (Figure 2D), and PDMS was poured in a 60-mm culture dish so as not to overflow the edge (Figure 2E). After leaving it at 25 \({}^{\circ}\)C for 9 h to cure the PDMS, the aluminum cylinder and culture dish were peeled off, and the molded PDMS taken out. Using a biopsy punch (BP-L20K, Kai, Tokyo, Japan), a 2-mm diameter hole was drilled at the intersection between the circular arc of the small recess structure and the Y-Y' part of the fabricated PDMS adapter, and at a linear small recess structure 2 mm away from the edge of the adapter, to serve as an inlet. Similar holes were made on each opposite side to form an outlet (Figure 2F,G). The completed sealed adapter was a cylindrical-shaped PDMS with a 2 mm wide, 500 \(\upmu\)m deep recess structure on a 2 mm thick bottom membrane, and an inlet and outlet with an inner diameter of 2 mm (Figure 2G). The fabricated CD-adapter had a very high transparency and was composed of PDMS only, and thus it has good oxygen permeability and a very suitable structure for cell culture. Furthermore, because the central membrane portion was a thin transparent membrane with a thickness of 2 mm, it was also possible to observe the culture dish with the adapter attached on it with a microscope (Figure 3A). The fabricated U-shaped and I-shaped structures are shown in Figure 3B,C, respectively. As shown in Figure 3D, a transparent framework made of acrylic resin was used as a holding jig to prevent liquid leakage. It has a circular shape so that a constant pressure can be applied to the side of the culture dish for tight sealing. In addition, the center of the culture jig contains a hole with a diameter of 10 mm through which cells can be observed using a microscope. The holding jig has a height of 5 mm and an outer diameter of 27.0 mm.