## 4 Discussion  
In the present study, all tested ECM substrates were successfully scaffolded on a fluoropolymer, and both HepG2 and MCF7 cell lines formed spheroids after culturing, without negatively affecting the cell population. The key to achieving this was the combination of the fluoropolymer and ECM dot pattern. Fluoropolymers are generally known to be difficult regarding adhesion; thus, many studies have attempted to improve adhesion by surface modification [78]. However, we showed that cells can adhere to the substrate via an ECM that can be detached easily by pipetting. The survival rate of the spheroids derived using our method was significantly lower than that of the cells in the 2D culture. The reason for this was speculated to be the  
Figure 4: Spheroids derived from HepG2 or MCF7 cells after culturing in 2D for 7 days on different ECM substrates. HepG2 cells seeded on Matrigel (**a1**), Matrix (**a2**), fibronectin (**a3**), vitronectin (**a4**), and collagen IV (**a5**). MCF7 cells seeded on Matrigel (**b1**), Matrix (**b2**), fibronectin (**b3**), vitronectin (**b4**), and collagen IV (**b5**). (**c**) Viability confirmation by trypan blue staining. Student \(t\)-test was performed to compare the survival rates between the 2D and spheroid (\(p\) = 0.01). \(p\) value < 0.05 was considered statistically significant. Data are expressed as means \(\pm\) standard deviations. The asterisks (*) indicate a statistically significant difference. (**d**) MCF7 on days 1 and 7 following the hanging-drop method. lack of space for cell growth inside the spheroid and the lack of nutrient circulation in the culture medium. For cancer spheroids, however, a slight decrease in survival are permitted; there are, accordingly, no disadvantages of our method. In addition, the hanging drop method could not produce large-sized spheroids in 7 days, but our method was effective in producing such large spheroids. It was also found to be more efficient because increasing the number of seeded cells could reduce the number of days required for spheroid formation (data not shown). These results suggest that our method can also be applied to spheroids derived from cancer cells. We assume that our method would be useful in cases where cells of interest are induced from pluripotent stem cells, such as iPSCs, to produce spheroids. This method involves the process of culturing in 2D for a period of time to proliferate and induce, and the process of creating spheroids to confirm the functionality of the cells of interest. Therefore, the first half of the experiment (about 7 days) was assumed to be the cell proliferation and differentiation stage and the second half was assumed to involve the production of spheroids. We assume that the greatest benefit of producing spheroids in this way is the elimination of chemical and physical stimuli. The cell line was passaged using an enzyme but was not used to produce spheroids. Since the cell line used here is passaged every 2-4 days, we minimized the effect of enzyme treatment on cells by assigning 7 days for colony formation. Thus, when increasing the seeding amount and attempting spheroidization in a short period of time, it is necessary to consider the effect of enzyme treatment. Fluoropolymers have a high biocompatibility and inertness due to their non-polarity and strong C-F bonds. They are, thus, a safe and non-toxic material for use in the living body. Their high inertness entails advantages, such as a low adhesion in vivo, while their hampered in vivo settling is a problem for biomaterials. Their symmetrical molecular structure makes them chemically resistant and stable against most chemicals, including acids, alkalis, and organic chemicals. However, they can react slightly with molten alkali metals and their solutions as well as fluorine and chlorine trifluoride at high temperatures. Therefore, the ECM used in this study was not expected to form strong chemical bonds. In addition, the non-polar molecular structure does not allow for electrical bonds, such as hydrogen bonds.