Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

Abstract

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

Keywords: assay validation; cell-based assays; oncology; patient-derived organoid culture.

Figure 1.

Establishment of patient-derived organoid cultures in 384-well format. (A, B) Patient-derived organoid samples were cultured and expanded (4 d in culture in A and 12 d in culture in B) in Matrigel droplets in 12-well plates. (C, D) Long-term cultures were stained for F-actin (phalloidin, red), Ki-67 (green), and DAPI (blue) and exhibited numerous budding structures, morphological integrity, and Ki-67–positive cells at the organoid surface. (E) Organoid samples were disaggregated into single-cell suspension and were seeded into 384-well plates. Compound treatment was initiated after a preculture period of 4 d, and cell viability was analyzed after a culture period covering two population doubling times. (F–I) Single cells were seeded into 384-well plates and subsequently formed three-dimensional organoid structures within 4 d of preculture. (J–M) Confocal image analysis of patient-derived organoid cultures in 384-well format stained for F-actin (phalloidin, red), Ki-67 (green), and DAPI (blue) illustrates the maximum intensity projection (J), an optical section of the surface (K), and the DAPI-negative luminal compartment of the center (L, M) of an organoid structure. Scale bars: A, B, F, H = 500 µm; C, D = 200 µm; G, I–M = 50 µm.

Figure 2.

Spatial uniformity assessment with optimized assay conditions. Patient-derived organoid samples in 384-well format were precultured for 4 d and subsequently treated with the vehicle control. (A, B, G). When plates were incubated without sealing membranes, plate edge effects were detected by higher raw signals of the relative luminescence units (RLU) in rows A and P and columns 1 and 24, resulting in a coefficient of variation (CV) of 18.97%. (C, D, G). By employing sealing membranes during the vehicle incubation period, edge effects were slightly reduced with a CV of 14.62%. (E, G) The use of the sealing membranes throughout the whole culture and treatment duration significantly reduced plate edge effects and showed spatial uniformity with a CV of 9.37%.

Figure 3.

Assay validation by plate uniformity study. The plate uniformity study was carried out over three independent runs consisting of two concentration-response curve (CRC) plates (CRC1 and CRC2), one plate with the maximum signal (Max) and one plate with the minimum signal (Min) for each run for the patient-derived organoid sample 250-MW-P-TF-01-03. (A, B) The raw signals of the relative luminescence units (RLU) of all 12 plates were plotted against the respective plate column (A) and visualized by plate heat maps (B). (C) The four-parameter logistic CRCs were plotted for each side (left and right) of the CRC plates for all three runs, and each color represents a plate row.

Figure 4.

Streamlined plate uniformity study for different patient-derived samples and replicate-experiment study. (A–C) The streamlined plate uniformity study was carried out over two independent runs consisting of one concentration-response curve (CRC) plate and one plate with the maximum signal (Max) for each run. (A, B) The raw signals of the relative luminescence units (RLU) of all four plates for each patient-derived sample (159-MB-P-TF-01-03, 327-MB-P-TF-01-03, and 364-CB-M-MF-01-04) were plotted against the respective plate column (A) and visualized by plate heat maps (B). (C) The four-parameter logistic CRCs were plotted for each side (left and right) of the CRC plates for the two runs for the different patient-derived samples, and each color represents a plate row. (E, F) The replicate-experiment study was carried out for one patient-derived sample (159-MB-P-TF-01-03) consisting of two independent runs with the same set of compounds. The minimum significant ratio (MSR), limits of agreement (LsA) for potency estimates and the correlation of the IC₅₀ values of both runs (E) as well as the minimum significant difference (MSD), limits of agreement for the difference (LsAd) for efficacy estimates, and the correlation of the maximum inhibition values of both runs (F) were calculated and visualized. The values for potency estimates were MSR = 2.72; LsA = 0.39 to 2.89; RLs = 0.89 to 1.27; MR = 1.06 (E). The values for efficacy estimates were MSD = 14.18; LsAd = −19.03 to 9.33; DLs = −7.41 to −2.30; MD = −4.85 (F). MD, mean difference; MR, mean ratio; DLs, difference limits (statistical limits of the mean difference); RLs, ratio limits (statistical limits of the mean ratio).