Volumetric time-course analysis of chemotherapeutic dose-response in 3D hydrogel embedded cancer spheroids using VitroPrime™ 3D Culture and Imaging Plates together with CYTOQUBE light-sheet microplate cytometer

Authors
Rick Cohen, Ph.D., John Huang, Ph.D., Mahomi Suzuki, Ph.D.

ABSTRACT

Predicting drug efficacy in oncological microphysiological systems (MPS) requires high-resolution longitudinal tracking of cell death pathways within dense 3D architectures. Traditional confocal methods struggle with light attenuation and phototoxicity during multi-day imaging. We present an example of a semi-automated HTS workflow using a system that (1) maintains the in vivo cancer phenotype by growing tumor cells using VitroGel® Glioblastoma (GBM) Xeno-
Free EMT Kit (2) uses VitroPrime™ 3D culture and imaging plates for a zero-disruption 3D cell culture workflow with “sample-locking” technology (3), harnesses the power of the CYTOQUBE, an automated light-sheet microplate cytometer capable of 4 color analysis in 21 minutes.

We performed time-course dose-response assays on U87 glioblastoma spheroids. Spheroids were cultured in 96-well VitroPrime™ 3D Culture and Imaging plates containing a 1.5 mm hydrogel layer. This ensured a comprehensive sampling of spheroids at varying depths, accounting for differential dye diffusion gradients between surface and core populations.

Cells were treated with a dose range of temozolomide and monitored over 72 hours using a 4-color multiplexed panel: Hoechst 33342 (total nuclei), CellROX Green (oxidative stress), CellEvent Caspase-3/7 Red (apoptosis), and SYTOX DeepRed (necrosis). Scanning of the wells and normalizing for total nuclei revealed distinct spatiotemporal waves of oxidative stress preceding caspase activation. The use of a synthetic, tunable matrix provided superior optical clarity
and consistent drug penetration compared to animal-derived scaffolds.

Quantitative volumetric analysis demonstrated a clear sigmoidal dose-response curve, with CYTOQUBE light-sheet Zyncscan technology providing significantly higher sensitivity for core-situated necrotic events than standard widefield methods. This methodology enables a standardized, high-throughput approach for evaluating the “method of kill” in 3D cancer models, facilitating more accurate lead optimization in drug development.

VitroPrime™ 3D Culture and Imaging Plate

VitroGel® Glioblastoma (GBM) Xeno-Free EMT Kit

CYTOQUBE Light-Sheet Microplate Cytometer