Category: Assay Development and Screening
A micropillar/microwell chip platform, which cultures cells three-dimensionally, is applied to a 3D cell-based multicolor assay for high-content screening (HCS). In previous studies, colonies cultured in the micropillar/microwell chip platform were simply stained to measure cell viability and calculate IC50 (single-parameter assay) in a high-throughput manner. However, they lacked the ability to give target-specific signals (multi-parameter assay) for better understanding of complex biological functions and mechanisms of drug action. To stain 3D-cultured colonies in alginate spots, we developed and optimized antibody staining procedures of a 3D cultured colony including fixing, permeabilizing, and blocking, in a high-throughput manner. As proof of concept, the phospho-EGFR (p-EGFR, highly expressed receptor protein in cancer), F-actin (one protein of actin cytoskeleton protein), and nucleus in a 3D cultured colony are stained and analyzed after treating 72 drugs. By comparing p-EGFR expression with F-actin and nucleus, the p-EGFR inhibition of the drugs are successfully identified in the 3D cultured colony. In addition to verifying the p-EGFR inhibition of the selected drugs, the p-EGFR expressions are measured using a western blot assay.
Sangyun Lee– graduate student, sungkyunkwan university, seoul, Seoul-t'ukpyolsi, Republic of Korea
seoul, Seoul-t'ukpyolsi, Republic of Korea
1. High-Throughput, High-Content Image Screening (HTS, HCS)
2. 3D patient cancer cell printing on a chip for disease modeling and personalized cancer therapy
3. High-Content Imaging (HCI) with 3D-cultured patient cells