Development of a 3D-optimized microplate enables confocal high content imaging with cell level resolution and the automation of immuno-staining methods for spheroid models

Homeostasis at the tissue level is maintained via finely orchestrated cell-cell and paracrine signaling which frequently involves several different cell types. Further, the cyto-architecture of tissues, including the organization and the ratio of tissue-specific cell types, plays an important role in maintaining normal tissue function and viability. Studying individual cell responses in the context of intact tissue is therefore critical to understanding the development, prevention, and reversal of human disease. To that end, we developed multi-cellular 3D liver, islet, and tumor microtissue models (a.k.a. spheroids), and an accompanying Akura™ 384 microplate technology that supports both spheroid production and high-resolution 3D imaging. The combined platform enables the cell-level analysis of tissues with added spatial resolution and architectural context. The platform is also scalable and automation-compatible making it ideal for studying tissue-level responses at early stages in the drug discovery pipeline.

Using pancreatic islet microtissues we examined the compatibility of the Akura™ 384 spheroid plate with confocal high-resolution imaging. Human islet microtissues, reconstituted from dissociated native islets, were labeled with two nuclear markers, DAPI (marker for all cells) and anti-NKX6.1 (beta cell-specific marker). Using a 3D nuclear colocalization assay requiring single cell resolution, we examined the relative contributions of the Akura™ 384 plates and a u-bottom spheroid plate to light attenuation, optical aberrations, and ability to perform accurate segmentation of the nuclei. Our results demonstrate that a continuous, flat, ultra-thin, transparent bottom significantly minimizes the refractive index mismatch and the chromatic registration issues observed with conventional u-bottom plates and enhances the overall speed and accuracy of the image acquisition.

Next the automation-compatibility of the Akura™ 384 plate was evaluated via the implementation of a fully automated fixation, permeabilization, immuno-staining, and tissue clearing method for tumor spheroids on an Opentrons OT-2 pipeting station. The spheroid model, comprised of GFP-expressing DLD-1 colorectal adenocarcinoma cells, was subjected to the automated overnight Fix-Perm-Stain-Clear method. The following day the plates were imaged on a Yokogawa CQ-1 high content analysis system and evaluated for preservation of spheroids, tissue clarity, and signal uniformity in 3 channels. Our results suggest that the unique well geometry, consisting of spheroid compartment and a contoured pipeting ledge, protects the microtissue from accidental aspiration and damage and enables fully automated processing and 3D imaging, consistent with high throughput workflows.

As the dependence on 3D models and high content imaging continues to expand, maximizing 3D image quality is imperative to the development of accurate and comprehensive spheroid measurements. Likewise, the implementation of reliable automated workflows is essential to the adoption of 3D models for screening applications. Innovations, such as the development of standardized 3D models and 3D-optimized microplates, may soon overcome the remaining obstacles limiting the broader utilization 3D cell models in early drug discovery.