High-Throughput Clonogenic Analysis of 3D-Cultured Patient-Derived Cells with a Micropillar and Microwell Chip

Abstract

A high-throughput clonogenic assay with a micropillar-microwell chip platform is proposed by using the colony area of glioblastoma multiforme (GBM) patient-derived cells (PDCs) from colony images. Unlike conventional cell lines, PDCs from the tumor are composed of heterogeneous cell populations, and some clonogenic populations form colonies during culture while the rest die off or remain unchanged, thus causing the diverse distribution of colony size. Therefore, area-based analysis of the total colonies is not sufficient to estimate total cell viability or toxicity responses. In this work, the average and standard deviation of an individual colony's area calculated from the colony images were used as indicators for cell clonogenicity and heterogeneity, respectively. Two parameters (the total and average area of colonies) were compared to draw the colony's growth curve and measure a doubling time and dose-response curve (IC). Based on both analyses of two PDCs, 464T PDCs show a higher heterogeneity and clonogenicity than 448T PDCs. The differences in the doubling time and the IC according to the analysis methods suggest that the average area of colonies, rather than their total area, is suitable for heterogeneous and clonogenic samples.

Drug Efficacy Comparison of 3D Forming and Preforming Sphere Models with a Micropillar and Microwell Chip Platform

“…As in our previous works, [10][11][12][13] the microwell chip was divided into 62 lines (60 drugs and 2 controls), and each line had 1 × 6 microwell arrays for replicates. The colony viability was measured from six replicates for each line.…”

“…Many successful cases have been reported, [7][8][9][10][11][12][13][14][15][16][17][18] including our previously developed assay system. [10][11][12][13] For example, one study developed a high-throughput clonogenic assay using a micropillar/microwell chip platform. The high-throughput clonogenic assay is a drug efficacy analysis method that quantitatively measured the 3D cell colony formation of glioblastoma multiforme (GBM) patient-derived cells (PDCs) according to various drugs.…”

“…The high-throughput clonogenic assay is a drug efficacy analysis method that quantitatively measured the 3D cell colony formation of glioblastoma multiforme (GBM) patient-derived cells (PDCs) according to various drugs. 13 It can determinate which drugs inhibit colony formation from a single cancer cell. Other papers [14][15][16][17][18] have successfully reported 3D cell culture models that showed the high resistance of the drug compared with the 2D monolayer cell culture model.…”

High-Dose Compound Heat Map for 3D-Cultured Glioblastoma Multiforme Cells in a Micropillar and Microwell Chip Platform

“…Previous high-dose heat map models using 2D cell-based high-throughput screening are well developed [ 9 , 10 ]. However, because 2D cell-based assay does not fully reflect in vivo microenvironments (cell-to-cell and cell-to-extracellular matrix interaction), a 3D cell-based assay was used to screen compounds [ 11 – 15 ], including our previously developed system [ 13 – 17 ]. Especially, 3D cultured astrocyte and GBMs show more in vivo like model [ 18 – 20 ].…”

“…Thus, assay based on 3D cultured astrocyte and GBMs with high-throughput manner may give new potential to screen GBM target agents. Our previous system [ 13 – 17 ] shows successfully data of 3D cell-based assays with high-throughput manner by comparing their own data with 2D cell-based assay [ 13 ], gene [ 14 ], and clinical data [ 17 ]. By applying the abovementioned quantitative 3D-cultured cell-assay platform, astrocytes and patient-derived GBM cells were 3D-cultured and screened to select the most represented compounds that were not cytotoxic to normal brain cells and were particularly efficient for patient-derived GBM cells.…”