Overview

We describe validation methods and data demonstrating the ability to kinetically image and quantify the growth, death and morphology of organoids embedded in Matrigel®. Using the Incucyte® Live-Cell Analysis System and associated lab tested protocols, automated label free quantification of organoid area, count and changes in organoid shape and darkness metrics provide insight into organoid differentiation and organoid response following perturbation.

Introduction

Organoid technologies are becoming vital in-vitro models for studying human development and disease, as they closely mimic real-life pathophysiology. However, to effectively utilize them in diverse research areas, it's essential to minimize variability and develop methods for imaging and measuring these intricate cell models. We describe simple, robust workflows for monitoring and automatically quantifying features, of organoids using real time live-cell analysis.

Methods

Matrigel® embedded organoid cultures in 96-well plates were imaged over time in an Incucyte® Live Cell System. Following perturbation, organoid growth and death were measured using Incucyte’s automated Organoid Software Analysis Module, which tracks changes organoid in size, number, organoid eccentricity and darkness. In 96-well plates, intestinal and hepatic organoid fragments were embedded in Matrigel® (50%) for 3 days prior to treatment with protein kinase inhibitor staurosporine (1 µM, STP).

Results

Label-free size and morphology metrics were used to distinguish between cytotoxic and cytostatic mechanisms of action of known chemotherapeutic compounds. STP, cisplatin (CIS, DNA synthesis inhibitor) or fluorouracil (5-FU, thymidylate synthetase inhibitor) exhibited concentration dependent inhibition of hepatic organoid growth, yielding IC50 values of 3 nM for STP, 9.7 µM for CIS and 0.78 µM for 5-FU. Differences between size and morphology readouts illustrated the cytostatic mechanism of 5-FU.

Conclusion

The Incucyte® Live-Cell Analysis System with the Incucyte® Organoid Analysis Software Module enables the study the growth or death of organoids, without the use of fluorescence. A proprietary brightfield image acquisition approach enables real-time kinetic imaging of 3D organoids embedded within a matrix (Matrigel®). This label-free method allows for real-time tracking and analysis of organoid morphologies and drug responses across a range of disease areas and organoid applications.