## 4 Discussion
Nevertheless, the cells were able to adhere to the ECM that was transferred onto the fluoropolymer (Figure 3a,c). Hydrophobic interactions [79] and the surface charge of the fluoropolymer [78] were reported as possible mechanisms underlying this adhesion. However, we believe that van der Waals forces are instead responsible because of the weak adhesive force that allowed cells to peel off over time regardless of the ECM type. During formation of the ECM dots, the ECM floating in the culture medium settled down due to gravity and gathered on the substrate surface after the gel-like protein molecules (matrix components) were stamped. As a result, the distance between the protein molecules and the fluoropolymer surface approached zero, and subsequently, the protein molecules adhered to the fluoropolymer surface via van der Waals forces. Van der Waals forces are extremely weak compared with chemical bonds and hydrogen bonds. Therefore, when the cells proliferated and the tension between them increased, the cells began to detach from the edges of the cell colonies and were easily peeled off from the ECM-coated fluoropolymer surface upon pipetting with a weak liquid flow. If the ECM indeed adheres via van der Waals forces, the question is why the fluoropolymer does not allow adhesion of other substances and is considered an inert material. This may be because the objects to which the fluoropolymer adheres are usually considered solids. The surfaces of solids possess microscopic irregularities, which are shown in Figure 5a. There are many gaps between the solid and the surface of the fluoropolymer. Because van der Waals forces are only generated when the distance is very short, the area ratio of the contact point to the entire interface is extremely small with solids; if the contact area/external force ratio is large, the adhesive force increases. Therefore, in many cases, it is impossible to obtain an attractive force sufficient to hold the entire solid against external forces. In contrast, when the object to be adhered is a liquid (Figure 5b), there is no gap,and the molecules are in close contact with each other. However, the molecules in the liquid move over long distances, and the van der Waals forces are not strong enough to overcome their kinetic energy and maintain the molecules at the interface. If the attractive forces are strong, the contact angle should be small, but this is not the case. Furthermore, because of the flow phenomenon, i.e., the shape of the entire liquid can freely change, it is not possible for the liquid to stay in place. In contrast, water (H\({}_{2}\)O) can remain on a glass surface because of the presence of a larger number of hydrogen bonds than in silicone oil. Moreover, when highly viscous silicone oil with extremely low polarity is dropped onto a fluoropolymer, the surface becomes wet easily, and the oil remains on the surface, even if it is physically wiped off. The ECM used in the present study [79; 80; 81; 82; 83; 84] is a gel with properties between those of a solid and liquid (Figure 5c); it can be deformed to the extent that no gaps are created between it and the fluoropolymer surface. We thus concluded that the protein molecules adhered to the fluoropolymer surface via van der Waals forces. In this study, the dimensions of the spheroids were too large compared to the dimensions of the 2D matrix dots (Figure 3e). The reason is considered to be that cell lines were used here. Seven days after seeding, it was observed that the number of cell in the colony exceeded that in the 2D confluent state and proliferate in the 3D layer. If the colonies were detached by pipetting before that, the spheroid size could be controlled. In the future, the proposed method will be employed to spheroids using stem cells, and in that case, such a problem could be automatically avoided. At that time, we expect that it will also be possible to induce all cells in a planar manner at the 2D culture stage, and then suspend them while maintaining the generated cell polarity to create 3D cells using our method.