3D cell subculturing pillar dish for pharmacogenetic analysis and high-throughput screening

Abstract

A pillar dishe for subculture of 3D cultured cells on hydrogel spots (Matrigel and alginate) have been developed. Cells cultured in 3D in an extracellular matrix (ECM) can retain their intrinsic properties, but cells cultured in 2D lose their intrinsic properties as the cells stick to the bottom of the well. Previously, cells and ECM spots were dispensed on a conventional culture dish for 3D cultivation. However, as the spot shape and location depended on user handling, pillars were added to the dish to realize uniform spot shape and stable subculture, supporting 3D cell culture-based high-throughput screening (HTS). Matrigel and alginate were used as ECMs during 6-passage subculture. The growth rate of lung cancer cell (A549) was higher on Matrigel than on alginate. Cancer cell was subcultured in three dimensions in the proposed pillar dish and used for drug screening and differential gene expression analysis. Interestingly, stemness markers, which are unique characteristics of lung cancer cells inducing drug resistance, were upregulated in 3D-subcultured cells compared with those in 2D-subcultured cells. Additionally, the PI3K/Akt/mTOR, VEGFR1/2, and Wnt pathways, which are promising therapeutic targets for lung cancer, were activated, showing high drug sensitivity under 3D-HTS using the 3D-subcultured cells.

Keywords: 3D cell culture; Cell culture dish; High-throughput screening; Micropillar and well chips.

Graphical abstract

Fig. 1

Schematic diagram of pharmacogenomic comparative analysis according to lung cancer cell culture model. A) Evenly dispensing and subculturing the cell/hydrogel mixtures are difficult on conventional cell culture dishes. B) The cell/hydrogel mixtures can be evenly dispensed, achieving high reproducibility of 3D cell subculture, on the proposed pillar dish. C) Single lung cancer cell line stock and subculture preparation. D) 3D-cultured lung cancer cells formed spheroids in the Matrigel dome, with high physiological relevance. E) 2D-cultured lung cancer cells were attached to the bottom of the dish, with low physiological relevance. F) Genomic analysis of lung cancer cells cultured under individual culture conditions. G) Anticancer drug sensitivity HTS analysis of lung cancer cells under individual culture conditions. Further, gene–drug association analysis was performed by comparing the results of genomic analysis and HTS anticancer drug sensitivity analysis.

Fig. 2

Measurement of the proliferation rate of subcultured lung cancer cells according to ECM. A) BF images of lung cancer cells cultured for 0–4 days according to ECM. B) Subculture proliferation rate of lung cancer cells according to ECM. The rate was approximately 645% on Matrigel and approximately 463% on alginate. The coefficient of variation (CV) values of 0.8% and 2.4% for the Matrigel and Alginate conditions, respectively, show high subculture performance.

Fig. 3

HTS anticancer drug sensitivity analysis results using micropillar/microwell chip. A) Scanning image of micropillar chip exposed to 72 compounds (including two DMSO controls). B) Comparative analysis of anticancer drug sensitivity according to cell culture model based on the relative cell viability value. C) Comparative analysis of anticancer drug sensitivity according to cell culture model based on the Z-score value.

Fig. 4

Transcriptome profiling according to lung cancer cell culture model. A, B) Volcano plot of differentially expressed genes showing the magnitude and significance of gene upregulation in 3D- and 2D-cultured lung cancer cells. C) GO analysis of hallmark genes enriched in 3D-cultured lung cancer cells. D) Analysis of hallmark genes enriched in 3D cultured lung cancer cells through differential gene expression analysis between lung cancer cell stocks and subculture models.

Fig. 5

Gene expression analysis of stemness markers related to increased drug resistance in 3D-cultured lung cancer cells. A) Violin plots indicating the activity level of stemness-related pathways using ssGSEA. B) Differences in transcript expression between 2D- and 3D-cultured lung cancer cells.

Fig. 6

Sensitive drug selection under 3D-HTS conditions and cross-validation through genomic analysis. A) Selection of drugs showing sensitive response under 3D-HTS conditions. B) Analysis of hallmark genes enriched in 3D-cultured lung cancer cells through differential gene expression analysis between 2D-HTS and 3D-HTS models. C) Violin plots indicating the activity levels of the PI3K/Akt/mTOR, Wnt, and VEGF pathways using ssGSEA.

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