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PMT and Image - Based Analysis of Hypoxia Induction using a 3D Spheroid Model

Monochromator, Image, and Cellular Analysis of Kinetic Hypoxia Induction in a 3D Liver Microtissue

Brad Larson - BioTek Instruments, Inc., Winooski, Vermont, USA
Stewart Hunt InSphero, Inc., Cambridge, Massachusetts, USA
Wini Luty - Enzo Life Sciences, Farmingdale, New York, USA
Peter Banks - BioTek Instruments, Inc., Winooski, Vermont, USA

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Introduction

Cell-based assays provide a wealth of drug screening information compared to their biochemical counterparts, and at a fraction of the cost and time seen with traditional animal models. It is estimated that half of the screens per formed for target validation and ADME-Tox now incorporate a cell-based format1. The evolution into live cell formats provides valuable data that may be missed in a single time point, lytic assay. In addition to a deeper understanding of cellular mechanics and responses with the fact that cells are still intact during the detection process, live cell assays offer increased flexibility through kinetic, or multiplexed analyses. A downfall of both these assay types is that cells grown on solid, flat substrates do not display the same morphology, and may not behave in the same manner, as those grown in vivo1.

Unlike ex vivo 2D cultured cells, those grown in vivo interact with nearby cells and the extracellular matrix (ECM) to form complex communication networks that control a number of cellular processes2. New 3D culture methods encourage cells to aggregate into clusters, thereby forming vital communication networks, and more closely mimicking in vivo structures. As these cell clusters, or spheroids, are quite small relative to the entire area of a microplate well, accurate PMT-based detection may be difficult. Imaging the spheroids, and incorporating cellular analysis instead of whole well analysis, may offer greater sensitivity.

Here, we investigate the impact of exposing cells cultured into a 3D microtissue to hypoxic atmospheric conditions. Kinetic monitoring was carried out by performing whole well monochromator-based reading and imaging over an 11 hour time period. Analysis of changes in whole-well and whole-image fluorescence intensity values, as well as changes specifically from cells making up the spheroid alone, via cellular analysis, demonstrate how the latter allows for a more accurate account of the final effect.

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