# Enhanced Drug Screening Efficacy in Zebrafish Using a Highly Oxygen-Permeable Culture Plate
## 4 Discussion  
This study highlights the substantial benefits of employing a highly oxygen-permeable culture plate, known as the InnoCellTM plate, for drug screening using zebrafish. Sufficient oxygen supply is crucial for embryonic development, as it is vital for metabolism, growth, and differentiation, especially in fast-developing organisms such as zebrafish. Hypoxic conditions can rapidly arise in traditional PS culture plates, negatively affecting cellular metabolism and viability [5; 6]. The innovative InnoCellTM plate, made from PMP, substantially enhances oxygen permeability compared with standard PS plates, thereby markedly improving cellular metabolism and function [11]. In this study, we found that the InnoCellTM plate demonstrated notably enhanced developmental outcomes in zebrafish embryos, including heart rate and body length, under both normal and oxygen-restricted conditions, compared with conventional PS plates (Figure 1). These results align with earlier findings in mammalian cell cultures and extend the positive effects of PMP materials on vertebrate embryogenesis and organism-level development. Notably, under oxygen-restricted (sealed) conditions, the advantages of the InnoCellTM plates were more notable. Embryos cultured on conventional PS plates exhibited marked developmental delays, including reduced body length, delayed yolk absorption, impaired pigmentation, and significantly lower hatching rates at higher densities (Figure 1E,G). Conversely, embryos in the InnoCellTM plate maintained relatively normal developmental profiles, suggesting that even limited oxygen transfer through the PMP base was sufficient to alleviate severe hypoxic stress and support normal developmental trajectories. Interestingly, under the same conditions (Figure 1C), embryos grown on PS plates unexpectedly  
Figure 6: Expression levels of _kdrl_ upon exposure to TKIs under “Sealing” conditions. **(A,B)** Relative mRNA expression levels of _kdrl_ at 96 h post-fertilization (hpf) exposed to 20 \(\mu\)L TKIs under “Sealing” conditions using the 96-well control **(A)** and the InnoCellTM **(B)** plates. **(C,D)** Relative mRNA expression levels of _kdrl_ at 96 hpf exposed to 15 \(\mu\)L TKIs under “Sealing” conditions using the 384-well control **(C)** and the InnoCellTM **(D)** plates. * \(p\) < 0.05, **_p_ < 0.01, *** \(p\) < 0.001, *** \(p\) < 0.0001, ns = not significant. \(n\) = 4. Data are presented as the mean \(\pm\) standard deviation (SD). TKIs, tyrosine kinase inhibitors; VPA, valpro acid. demonstrated elevated heart rates in almost all medium volumes. This may reflect a transient compensatory response to acute hypoxia, as previously reported in zebrafish embryos [23,24]. Hypoxia can trigger tachycardia to enhance oxygen delivery before cardiac function declines. In contrast, embryos in the InnoCell(tm) plate maintained stable heart rates, indicating adequate oxygen availability. Collectively, these results emphasize the hypoxia-induced physiological stress in PS plates and the advantages of utilizing oxygen-permeable systems, such as the InnoCell(tm) plate. Moreover, these findings highlight the critical importance of oxygen availability during zebrafish development and demonstrate that PMP plates with high oxygen permeability offer substantial benefits under difficult conditions, such as high embryo densities or sealed cultures. This technology enhances the assay consistency and has considerable potential to improve the reliability of zebrafish-based drug screening. In studies using 96-well plates (Figure 2), the InnoCell(tm) plate demonstrated superior support for the development of zebrafish embryos compared with traditional control plates across various medium volumes. Embryos grown in the InnoCell(tm) plate displayed notably higher heart rates and increased body lengths, especially at medium volumes ranging from 50 to 200 \(\upmu\)L per well, under both sealed and unsealed conditions.