# Enhanced Drug Screening Efficacy in Zebrafish Using a Highly Oxygen-Permeable Culture Plate
## 3 Results
### Developmental Assessment Using 96-Well Plates  
A 96-well plate was used for drug screening in zebrafish. Initially, we assessed the effectiveness of the InnoCellTM plate for embryo development and determined the minimum culture medium volume required. According to the guidelines outlined in The Zebrafish Book for nurturing zebrafish larvae, a density of 25-50 embryos per 100 mL (4000 \(\upmu\)L/fish) should be maintained [22]. Consequently, we incorporated a \"Petri dish\" condition as the standard control environment. Embryos were cultured individually in wells of both control and InnoCellTM plates with varying liquid volumes. Figure 2B illustrates that zebrafish in the InnoCellTM plate had significantly higher heart rates, reaching 190 \(\pm\) 2 bpm, compared to those of embryos on the control plate (179 \(\pm\) 6 bpm; \(p\)! 0.05) at a volume of 50 \(\upmu\)L per well per fish. Additionally, at a volume of 200 \(\upmu\)L per well per fish without sealing, embryo heart rates were 175 \(\pm\) 2 and 166 \(\pm\) 5 bpm for the InnoCellTM and control plates, respectively. At volumes of 30 and 40 \(\upmu\)L/well/fish under \"Sealing\" conditions, embryos cultured in InnoCellTM plates had higher average heart rates than those cultured on control plates (\(p\)! 0.05; Figure 2C). Similar trends were observed for body length measurements at 72 hpf (Figure 2D,E). Embryos cultured in the InnoCellTM plate exhibited a notable increase in body length at volumes of 15, 10, 100, and 200 \(\upmu\)L/well/fish without sealing compared with those cultured in the control plate (Figure 2D). Additionally, an increase in embryo body length was observed at 20, 40, 100, and 200 \(\upmu\)L/well/fish under sealed conditions in the InnoCellTM plate compared with that in the control plate (Figure 2E). Although 10,  
Figure 1: Evaluation of the zebrafish embryo development cultured in the 24-well control and InnoCellTM plates at different densities. (**A**) Experimental design and schema. (**B,C**) Heart rate measurements at 56 h post-fertilization (hpf) in embryos cultured in control and InnoCellTM plates. (**B**) “No sealing” condition; (**C**) “Sealing” condition. (**D,E**) Body length at 72 hpf cultured in the control and InnoCellTM plates under “No sealing” (**D**) and “Sealing” (**E**) conditions. (**F,G**) Representative images of embryos at 72 hpf cultured in the control and InnoCellTM plates at different densities under “No sealing” (**F**) and “Sealing” (**G**) conditions. * \(p\)! 0.05, ** \(p\)! 0.01, *** \(p\)! 0.001, \(n\) = 5. Data are presented as the mean \(\pm\) standard deviation (SD).  
15, 20, and 30 \(\upmu\)L/well/fish under \"No sealing\" conditions did not induce any issues in embryo development, a three-day culture resulted in notable evaporation. Accordingly, we identified \(\geq\)40 \(\upmu\)L/well/fish as the minimum volume for normal embryo development under \"No sealing\" conditions using the 96-well InnoCell\({}^{\text{TM}}\) plate. Based on body length results at 72 hpf, \(>\)20 \(\upmu\)L/well/fish was identified as the optimal minimum volume for embryo development under \"Sealing\" conditions using the 96-well InnoCell\({}^{\text{TM}}\) plate.