Abstract
Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis.
Highlights
The high attrition rate of drugs in clinical trials[1] is motivating a paradigm shift in preclinical drug discovery and development from traditional two-dimensional (2D) monolayer cell cultures to three-dimensional (3D) cell cultures and tissue models[2,3]
T47D and U87 spheroids were grown in 384-well round-bottom ultra-low attachment (ULA) plates and, for initial experiments, fixed at 3 days in vitro (DIV)
As expected, imaging of fixed, Hoechst-counterstained spheroids in phosphate-buffered saline (PBS) demonstrated that light scattering in the 3D samples prevented resolution of individual nuclei past several cell layers (Fig. 1b,c)
Summary
The high attrition rate of drugs in clinical trials[1] is motivating a paradigm shift in preclinical drug discovery and development from traditional two-dimensional (2D) monolayer cell cultures to three-dimensional (3D) cell cultures and tissue models[2,3]. High-throughput image analysis methods for spheroids generally rely on whole-spheroid fluorescent analysis of traditional live/dead stains[11,12,13], or brightfield analysis of spheroid size as a measure of compound cytotoxicity[2,13,14,15,16] and matrix invasion as an indicator of drug antimetastatic effects[15] While these analysis methods provide useful information, they do not provide detailed biological or positional information of cells within spheroids. The ability to resolve and segment individual cell nuclei in 3D culture models is a necessary prerequisite for performing high spatial resolution measurements and quantitation of microenvironment effects. We demonstrate utility of this protocol by segmenting spheroid nuclei, identifying fluorescent subpopulation percentages, and assessing clearing and segmentation success This workflow has the ability to change the field of 3D high-throughput analysis by enabling the acquisition of nuclear resolution information in 3D samples
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