Name: Video: Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging
Uploaded: 2024-01-05T14:20:00
Description: 88 Views. Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging

plating intact patient-derived organoids and setting up imaging parameters for multiplexed live-cell imaging

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are rapidly emerging as a robust model system to study cancer development and therapeutic responses. PDOs are three-dimensional (3D) cell culture systems that recapitulate the complex genomic profile and architecture of the primary tumor1,2. Unlike traditional two-dimensional (2D) cultures of immortalized cancer cell lines, PDOs capture and maintain intratumoral heterogeneity3,4, making them a valuable tool for both mechanistic and translational research. Although PDOs are becoming an increasingly popular model system, commercially available reagents and analysis methods for cellular effects that are compatible with PDO cultures are limited.
The lack of robust methods to analyze subtle changes in treatment response hinders clinical translation. The gold standard cell health reagent in 3D cultures, CellTiter-Glo 3D, utilizes ATP levels as a determinant of cell viability5,6. While this reagent is useful for endpoint assays, there are several caveats, most notably the inability to use samples for other purposes after completion of the assay.
Live-cell imaging is a sophisticated form of kinetic microscopy that, when combined with fluorescent reagents, has the capacity to quantify a variety of cell health readouts within PDO models, including apoptosis7,8,9 and cytotoxicity10. Indeed, live-cell imaging has been integral to high throughput screening of compounds in 2D platforms11,12. Systems such as the Incucyte have made the technology affordable and thus accessible to research groups in a variety of settings. However, the application of these systems to analyze 3D cultures is still in its infancy.
Herein, we describe a method to assess drug response in PDO models of cancer using multiplexed live-cell imaging (Figure 1). Through analysis of bright field images, changes in PDO&#160;size and morphology can be kinetically monitored. Furthermore, cellular processes can be simultaneously quantified over time using fluorescent reagents, such as Annexin V Red Dye for apoptosis and Cytotox Green Dye for cytotoxicity. The methods presented are optimized for the Cytation 5 live-cell imaging system, but this protocol may be adapted across different live-cell imaging platforms.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging

plating-intact-patient-derived-organoids-setting-up-imaging

Research

JoVE Journal

Cancer Research

88 Views. Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging

Video: Plating Intact Patient-Derived Organoids and Setting Up Imaging Parameters for Multiplexed Live-Cell Imaging

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...