# Enhanced Drug Screening Efficacy in Zebrafish Using a Highly Oxygen-Permeable Culture Plate
## 4 Discussion
Moreover, drug screening tests using TKIs demonstrated the effectiveness of the InnoCellTM plate in identifying antiangiogenic effects in zebrafish embryos compared with the traditional PS plates. In both 96- and 384-well formats, the InnoCellTM plate enabled the accurate evaluation of ISV formation and vascular gene expression (_kdrl_), even with reduced medium volumes and under oxygen-limited conditions (\"Sealing\"). Exposure to sorafenib and axitinib substantially inhibited ISVs, with consistent suppression observed on the InnoCellTM plate, particularly at lower volumes. Although SU4312 demonstrated less consistent effects on ISV morphology, it substantially reduced _kdrl_ mRNA levels, indicating that molecular responses can occur before or independently of visible vascular changes. This finding is consistent with previous studies that identified _kdrl_ as a sensitive early marker of antiangiogenic drug activity in zebrafish models [33; 34]. These results underscore the need to incorporate both phenotypic and molecular analyses to assess drug efficacy in zebrafish. Relying solely on vessel counts may lead to an underestimation of the biological activity of certain compounds, whereas combining morphological and gene expression evaluations would offer a more comprehensive assessment. Additionally, the data revealed that the high oxygen permeability of the InnoCellTM plate maintained assay sensitivity even under challenging conditions, such as reduced volume or sealing, which are crucial for high-throughput screening. Overall, the InnoCellTM platform enhances assay consistency, sensitivity, and scalability and advances zebrafish-based pharmacological assessment. To acknowledge the limitations of this study and inform future research, several important considerations are worth noting. First, although this study focused primarily on PS and PMP plates, future investigations comparing other materials, such as glass, polycarbonate, or cyclic olefin polymer, would be valuable. Differences in oxygen permeability across materials may significantly influence zebrafish development; however, direct comparative data remain limited. Expanding these comparisons could help establish more standardized criteria for plate selection in zebrafish-based assays. Second, this study evaluated developmental parameters up to 72-96 hpf, including the post-hatching stage (typically 48-56 hpf), which is critical for early phenotypic assessment. Due to size constraints, high-throughput formats such as 96- and 384-well plates are generally suitable only for embryonic and early larval stages. Larger formats (e.g., 6-well plates) may enable longer-term evaluation through the juvenile stage, although applying such systems to adult zebrafish remains technically challenging. Third, while this research focused on TKIs, initial findings suggest that the InnoCellTM plate is also effective for the reliable screening of other drug categories, including nephrotoxic and metabolic agents. Upcoming research will investigate its wider applicability in drug discovery across multiple classes and in toxicology tests. Finally, further evaluation using diverse genetic backgrounds, including mutant and transgenic lines, as well as at later developmental stages, will be essential for assessing its broader applicability.