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Abstract
Various three-dimensional (3D) cell culture methods have been developed to implement tumor models
similar to in vivo. However, the conventional 3D cell culture method has limitations such as difculty
in using an extracellular matrix (ECM), low experimental reproducibility, complex 3D cell culture
protocol, and difculty in applying to high array plates such as 96- or 384-plates. Therefore, detailed
protocols related to robust 3D-aggregated spheroid model (3D-ASM) production were optimized and
proposed. A specially designed wet chamber was used to implement 3D-ASM using the hepatocellular
carcinoma (HCC) cell lines, and the conditions were established for the icing step to aggregate the
cells in one place and optimized ECM gelation step. Immunofuorescence (IF) staining is mainly used
to simultaneously analyze drug efcacy and changes in drug-target biomarkers. By applying the IF
staining method to the 3D-ASM model, confocal microscopy imaging and 3D deconvolution image
analysis were also successfully performed. Through a comparative study of drug response with
conventional 2D-high throughput screening (HTS), the 3D-HTS showed a more comprehensive range
of drug efcacy analyses for HCC cell lines and enabled selective drug efcacy analysis for the FDAapproved drug sorafenib.
This suggests that increased drug resistance under 3D-HTS conditions does not reduce the analytical discrimination of drug efcacy,
also drug efcacy can be analyzed more selectively compared to the conventional 2D-HTS assay. Therefore, the 3D-HTS-based drug efcacy
analysis method using an automated 3D-cell spotter/scanner, 384-pillar plate/wet chamber, and the
proposed 3D-ASM fabrication protocol is a very suitable platform for analyzing target drug efcacy in HCC cells.