부산대학교 도서관

Establishment of drug testing of patient-derived cancer cells (PDCs) in physiologically relevant 3-dimensional (3D) culture is central for drug discovery and cancer research, as well as for functional precision medicine. Here, we describe the detailed protocol allowing the 3D drug testing of PDCs – or any type of cells of interest – in Matrigel in 384-well plate format using automation. We also provide an alternative protocol, which does not require supporting matrices. The cancer tissue is obtained directly from clinics (after surgery or biopsy) and processed into single cell suspension. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the PDCs directly after cancer cell isolation from tissue or on cells expanded for a few passages. In the 3D-DSRT assay, the PDCs are plated in 384-well plates in Matrigel, grown as spheroids, and treated with compounds of interest for 72 h. The cell viability is directly measured using a luminescence-based assay. Alternatively, prior to the cell viability measurement, drug-treated cells can be directly subjected to automated high-content bright field imaging or stained for fluorescence (live) cell microscopy for further image analysis. This is followed by the quality control and data analysis. The 3D-DSRT can be performed within a 1–3-week timeframe of the clinical sampling of cancer tissue, depending on the amount of the obtained tissue, growth rate of cancer cells, and the number of drugs being tested. The 3D-DSRT method can be flexibly modified, e.g., to be carried out with or without supporting matrices with U-bottom 384-well plates when appropriate for the PDCs or other cell models used.