Abstract 3084: Label-free, real-time live cell assays for 3D organoids embedded in Matrigel®
Authors: Jackson Tim R. , Oliver Miniver , Appledorn Daniel , Dale Tim , Barnes Kalpana 
Published: June 2022

Abstract
Organoid technologies are increasingly being used as in-vitro models of human development and disease as they exhibit structural, morphogenetic, and functional properties that recapitulate in vivo pathophysiology. To successfully use these models across a variety of research disciplines and applications, approaches that reduce variability and technology pipelines to image & quantify these complex cell models are required. Here, we demonstrate simple, robust workflows for monitoring and automatically quantifying features, such as morphology, growth and death of organoids using real time live cell analysis. To quantitatively optimize and characterize organoid cultures in-vitro, mouse hepatic, intestinal and pancreatic organoids were embedded in Matrigel® domes (50% or 100%) in 24-well plates and imaged over time in an Incucyte® Live Cell System. Organoid growth, differentiation, and maturation was measured using Incucyte’ s automated Organoid Software Analysis Module, which tracks changes in size and morphology. Integrated metrics enabled objective determination of cell-type specific growth conditions and passaging regimes. To illustrate the utility of the Incucyte® Organoid Analysis Software Module to track organoid growth and death 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). Vehicle treated organoids increased in size (10-fold; intestinal or 3-fold; hepatic) over time while marked reduction was observed in the presence of STP. Using label-free size and morphology metrics we could distinguish between cytotoxic and cytostatic mechanisms of action (MoA) 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. Whilst attenuation of size was observed across all compounds, increases in eccentricity and darkness indictive of 3D structure disruption and cell death respectively were only observed in CIS and STP-treated organoids (cytotoxic MoA). Differences between the size and morphology readouts illustrated the cytostatic mechanism of 5-FU. Use of this approach was extended to visualize and quantify CFTR function. Following forskolin stimulation, a concentration-dependent increase in intestinal organoid size was observed. In the presence of CFTR inhibitor CFTRinh-172 the maximal response was reduced by &gt;50% (~150% at 10 µM) illustrating that swelling was CFTR-dependent. These data demonstrate the capability to kinetically visualize and quantify distinct organoid morphologies, assess drug-induced cellular changes label-free and illustrates the amenability of this approach across a range of disease areas.
Citation Format: Tim R. Jackson, Miniver Oliver, Daniel Appledorn, Tim Dale, Kalpana Barnes. Label-free, real-time live cell assays for 3D organoids embedded in Matrigel® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3084.