ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids

Abstract

Source: Higa, A. et al. High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids. J. Vis. Exp. (2021)

In this video, we demonstrate a rapid, single-addition assay to assess cell viability in patient-derived organoids upon treatment with a test agent using intracellular ATP levels as a marker for cellular metabolic activity. The intracellular ATP levels are measured using the luciferase/luciferin reaction, where the emitted light intensity indicates the cellular viability and, in turn, the test agent's growth-inhibitory potency.

Protocol

1. Growth Inhibition High-Throughput Screening (HTS)

NOTE: The growth inhibitory activity of anticancer agents against patient-derived tumor organoids (PDOs) is evaluated by measuring the intracellular ATP content, as shown in Figure 1. This step is performed using a commercially available cell viability assay kit (see Table of Materials).

1. On day 0, culture the PDOs (e.g., RLUN007) in flasks until adequate numbers of cell clusters are available for the assay. One day before seeding, transfer the PDO suspension from a 75 cm² flask to a 15 mL tube and centrifuge at 200 x g for 2 min to measure the PDO pellet volume. Then, resuspend the PDO pellet in 15 mL of fresh medium and transfer it back to a 75 cm² flask. Culture the cells at 37 °C in 5% CO₂.
   NOTE: The required PDO pellet volume depends on each PDO's dilution rate and the number of 384-well plates used for the assay. For RLUN007, a 200 µL of cell pellet volume is necessary for seeding into ten 384-well plates.
2. On day 1 (24 h after changing the medium), mince the PDOs using cell fragmentation and dispersion equipment (see Table of Materials) with a filter holder containing a 70 µm mesh filter. Then, dilute 15 mL of the PDO suspension by 10x. Seed 40 µL of the PDO suspension into 384-well ultra-low attachment spheroid (round-bottom) microplates (see Table of Materials) using a cell suspension dispenser (see Table of Materials).
   NOTE: For mincing cell clusters, it is recommended to use the commercially available cell fragmentation and dispersion equipment (see Table of Materials).
3. At 24 h after seeding (day 2), treat the PDOs with 0.04 µL of test agent solutions at final concentration ranges of 20 µM to 1.0 nM (10 serial dilutions) using a liquid handler (see Table of Materials).
4. On day 8 (144 h after test substance treatment), add intracellular ATP measuring reagent to the test wells. Mix the plates using a mixer and incubate for 10 min at 25 °C. Measure the intracellular ATP content as luminescence using a plate reader (see Table of Materials).
5. To calculate the cell viability, divide the quantity of ATP in the test wells by that in the control wells containing the vehicle, with the background subtracted. To calculate the growth rate over 6 days, divide the quantity of ATP in the vehicle control wells by that in the vehicle control wells 24 h after seeding.
6. Calculate the 50% inhibitory concentration (IC₅₀) and area under the curve (AUC) values from the dose-response curves using biological data analysis software (see Table of Materials). The Z factor is a dimensionless parameter that ranges from 1 (infinite separation) to <0, defined as Z = 1 - (3σc+ + 3σc-) / |µc+ - µc-|, where σc+, σc-, µc+, and µc- are the standard deviations (σ) and averages (µ) of the high (c+) and low (c−) controls, respectively.

Results

Figure 1: Summary of the protocol used to create a high-throughput assay system using 384-well microplates.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
384-well Ultra-Low Attachment Spheroid Microplate	Corning	4516	Plates for HTS
Cancer Cell Expansion Media plus	Fujifilm Wako Pure Chemical	032-25745	Medium for F-PDO
CellPet FT	JTEC	-	Cell fragmentation and dispersion equipment
CellTiter-Glo 3D Cell Viability Assay	Promega	G9683	Cell viability luminescent assay, intracellular ATP measuring reagent
Echo 555	Labcyte	-	Liquid handler
EnSpire	PerkinElmer	-	Plate reader
Morphit software, version 6.0	The Edge Software Consultancy		Biological data analysis software
Multidrop Combi	ThermoFisher Scientific	5840300	Cell suspension dispenser
RLUN007	Fujifilm Wako Pure Chemical or Summit Pharmaceuticals International		Lung tumor derived F-PDO
Ultra-Low Attachment 75 cm² Flask	Corning	3814	Culture flask for PDO