# Enhanced Drug Screening Efficacy in Zebrafish Using a Highly Oxygen-Permeable Culture Plate
## 3 Results
### Application for Drug Screening  
Given the successful performance of the InnoCellTM plate in supporting zebrafish embryo development, we explored its effectiveness in drug screening applications. Antiangiogenic receptor TKIs were selected owing to their well-characterized and quantifiable biological effects in zebrafish models. The TKIs used in this study are listed in Table 1.  
Body length was selected as the reference parameter for assessing minimum volume because it encapsulates cumulative development over a span of 72 h. Conversely, heart rate \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Drug** & **CAS No.** & \begin{tabular}{c} **Molecular** \\ **Weight (Da)** \\ \end{tabular} & **Targets** & \begin{tabular}{c} **Exposure Concentration** \\ **(\(\mu\)M)** \\ \end{tabular} \\ \hline PTK787 & 212141-51-0 & 419.73 & \begin{tabular}{c} VEGFR2, VEGFR-1, \\ PDGF, Flt-4, c-Kit \\ \end{tabular} & 0.15 \\ SU5416 & 204005-46-9 & 238.29 & \begin{tabular}{c} VEGFR2, PDGFR, Fit-1, \\ Flt-4, c-Kit \\ \end{tabular} & 2.5 \\ SU4312 & 5812-07-7 & 264.3 & \begin{tabular}{c} VEGFR2, PDGFR, EGFR, \\ HER-2, IGF \\ \end{tabular} & 6 \\ Sorafenib & 284461-73-0 & 464.82 & \begin{tabular}{c} VEGFR, PDGFR, FGFR1, \\ KIT, RAF \\ \end{tabular} & 1.5 \\ Axitinib & 319460-85-0 & 386.47 & \
\begin{tabular}{c} VEGFR1, VEGFR, \\ VEGFR3, PDGFR, c-Kit \\ \end{tabular} & 0.5 \\ Valproic acid & 99-66-1 & 144.21 &
\begin{tabular}{c} HDAC1 \\ \end{tabular} & 5 \\ \hline \hline \end{tabular}  
* VEGFR, vascular endothelial growth factor receptor; PDGF, platelet-derived growth factor; Flt-4, Fms-like tyrosine kinase 4; c-Kit, mast/stem cell growth factor receptor Kit; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; HER-2, human epidermal growth factor receptor 2; IGF; insulin-like growth factor; FGFR, fibroblast growth factor receptor 1; RAF, rapidly accelerated fibrosarcoma; HDAC1, histone deacetylase 1.  
\end{table} 
Table 1: Properties of tyrosine kinase inhibitors. Figure 3: Evaluation of the zebrafish embryo development cultured in the 384-well control and InnoCellTM plates at different volumes. (**A,B**) Heart rate measurement at 56 h post-fertilization (hpf) cultured in the control and InnoCellTM plates under “No sealing” (**A**) and “Sealing” (**B**) conditions. (**C,D**) Body length at 72 hpf cultured in the control and InnoCellTM plates under “No sealing” (**C**) and “Sealing” (**D**) conditions. * \(p<0.05\), **\(p<0.01\), *** \(p<0.001\), \(n=4\)–8. Data are presented as the mean \(\pm\) standard deviation (SD). is more reactive to short-term stress and may not consistently serve as a reliable indicator of overall viability. For 96-well plates, the minimum and maximum volume selected were 40 and 100 \(\upmu\)L/well/fish, respectively, under the \"No sealing\" condition; for the \"Sealing\" condition, the minimum and maximum volumes selected were 20 and 100 \(\upmu\)L/well/fish, respectively. After drug exposure from 24 to 96 hpf, the zebrafish were anesthetized, and GFP fluorescent images were obtained to enumerate ISVs (Figure 4A). Embryos exposed to PTK787, SU5416, SU4312, or the negative control VPA showed no significant differences in the number of ISVs between the control and InnoCell\({}^{\text{TM}}\) plates, regardless of oxygen conditions and drug volume. Exposure to sorafenib and axitinib exerted strong angiogenic effects under almost all conditions. Embryos exposed to sorafenib showed a decreasing trend in the number of ISVs when cultured at 40 \(\upmu\)L/well/fish in InnoCell\({}^{\text{TM}}\) plates under the \"No sealing\" condition (\(p\) = 0.09; Figure 4B), although no differences were observed between the InnoCell\({}^{\text{TM}}\) and control plates at 100 \(\upmu\)L/well/fish (Figure 4C). Conversely, embryos cultured at 20 \(\upmu\)L/well/fish in the InnoCell\({}^{\text{TM}}\) plate exhibited a significantly lower number of ISVs than those cultures in control plates under \"Sealing\" conditions (\(p\) < 0.05; Figure 4D).