# Enhanced Drug Screening Efficacy in Zebrafish Using a Highly Oxygen-Permeable Culture Plate
## 4 Discussion
Remarkably, the InnoCell(tm) plate supported normal development even at lower volumes, down to 40 \(\upmu\)L per well without sealing and 20 \(\upmu\)L per well with sealing. In contrast, PS plates necessitated larger volumes to avoid developmental delays. This benefit is likely attributable to the improved oxygen permeability of the InnoCell(tm) plate, which compensates for the smaller medium volumes by ensuring adequate oxygen delivery. Determining the optimal minimum medium volume in 96-well plates is essential for balancing cost, efficiency, and embryo health. If the volume is extremely low, oxygen depletion, nutrient deficiency, and pH changes can hinder development [5]. Zebrafish embryos can reportedly withstand oxygen levels as low as about 3.33 mg/L without major developmental issues; however, a drop below this level may lead to stunted growth or death [25]. Moreover, evaporation in small volumes can concentrate substances, thereby increasing the risk of toxicity. Generally, medium volumes of 100-200 \(\upmu\)L per well are employed in zebrafish drug screening to ensure adequate oxygen and nutrients [26, 27, 28]. However, certain protocols allow this volume to be reduced to 50 or 70 \(\upmu\)L per well [29, 30], which can height the risk of hypoxia and evaporation, especially in PS plates [31]. Based on our findings, the InnoCell(tm) plate facilitates normal development even at volumes as low as 20-40 \(\upmu\)L per well, highlighting its potential for economical, high-throughput screening. This has substantial implications for drug screening processes, given that reducing the medium volume can decrease reagent expenses and increase screening efficiency. To further minimize evaporation-related artifacts at such low volumes, particularly under unsealed conditions, we recommend using only the inner wells and avoiding the outermost edge wells. This strategy is commonly used in high-throughput screening to mitigate edge effects and maintain consistent incubation conditions. Furthermore, the InnoCell(tm) plate provides a more stable and physiologically relevant environment, thereby enhancing the reliability and consistency of zebrafish-based assays, even in high-throughput scenarios. Our findings underscore the importance of integrating plate design with volume optimization to enhance zebrafish drug screening systems. The 96-well plates are commonly employed in zebrafish drug screening owing to their appropriate size and ease of use, whereas the 384-well plates are less frequently used because of technical difficulties, such as smaller wells, limited medium volume, and evaporation, which can affect embryo survival and data integrity [20]. Nonetheless, 384-well plates have notable benefits, including lower reagent costs, increased throughput, and compatibility with automated imaging systems, making them a promising option for large-scale chemical or genetic screening [18; 19; 32]. In our study involving a 384-well plate setup (Figure 3), the InnoCellTM plate exhibited distinct advantages over the standard PS plates. Embryos cultured in the InnoCellTM plate maintained substantially higher heart rates and greater body lengths, even when the medium volume was reduced to 20 \(\mu\)L per well without sealing and 15 \(\mu\)L per well with \"Sealing\" conditions that typically impede development in conventional plates. This is likely due to the high oxygen permeability of the PMP material, which helps overcome the oxygen constraints frequently encountered in the 384-well plate setup. Notably, this feature allows for a substantial reduction in medium volume without impacting embryo viability or assay sensitivity, which is crucial for high-throughput screening. These findings emphasize the potential of the InnoCellTM plate to enhance screening efficiency, cost-effectiveness, and data reproducibility in automated zebrafish-based systems.