Microarray Embedding/Sectioning for Parallel Analysis of 3D Cell Spheroids

Abstract

Three-dimensional cell spheroid models can be used to predict the effect of drugs and therapeutics and to model tissue development and regeneration. The utility of these models is enhanced by high throughput 3D spheroid culture technologies allowing researchers to efficiently culture numerous spheroids under varied experimental conditions. Detailed analysis of high throughput spheroid culture is much less efficient and generally limited to narrow outputs, such as metabolic viability. We describe a microarray approach that makes traditional histological embedding/sectioning/staining feasible for large 3D cell spheroid sample sets. Detailed methodology to apply this technology is provided. Analysis of the technique validates the potential for efficient histological analysis of up to 96 spheroids in parallel. By integrating high throughput 3D spheroid culture technologies with advanced immunohistochemical techniques, this approach will allow researchers to efficiently probe expression of multiple biomarkers with spatial localization within 3D structures. Quantitative comparison of staining will have improved inter- and intra-experimental reproducibility as multiple samples are collectively processed, stained, and imaged on a single slide.

Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture

“…Such tumor spheroid disaggregation on removal from ULA wells has been reported by other investigators. 30,31 It is unclear why this occurs, but it may be caused by differences in extracellular matrix deposition or by the intercellular adhesions between tumor cells. Ivanov and Grabowska 30 have cleverly designed agarose molds that do not require the tumor spheroids to be removed from the wells for histological embedding, sectioning, or staining; instead, spheroid tissue microarrays are created, allowing for high-throughput analysis of large 3D tumor cell spheroid sample sets.…”

The Application of the Tissue Microarray (TMA) Technology to Analyze Cerebral Organoids

“…Another technical problem encountered is the settling of the organoids in different planes during processing, causing inefficient evaluation of a histological section containing an entire collection of samples. This problem can partially be resolved using precasted agarose molds 34,35 where individual spheroids are embedded in cryomolds or individual paraffin blocks, using a manual microarrayer, in such a way as to align the paraffin array block to the cutting plane of the microtome. These precasted molds are very useful, but their main limitation is that all the organoids or spheroids need to have the same dimension (size and shape), which is not always possible.…”

“…33 Here, we describe how we used the TMA technology to study cerebral organoids starting from hiPSC differentiated into neurons and describe an alternative approach to previously published methods. 34,35 Generated organoids grown in different conditions and for different lengths of time were resuspended in low-melting agarose and arrayed using a semiautomatic tissue instrument with a proprietary software enabling sample deposition and condition tracing. The TMA technology faithfully captured cell morphology variations and detected stage-specific biomarkers.…”

4D‐Printed Transformable Tube Array for High‐Throughput 3D Cell Culture and Histology

“…[ 14,15 ] Furthermore, this demanding process has to be repeated for each specimen when a large number of 3D culture assays are to be investigated. There have been some efforts to speed up histology analysis using specially designed arrays, [ 16–19 ] but they are not compatible with standard culture devices such as multi‐well plates, thus still requiring harvesting and transferring steps. These painfully slow and labor‐intensive histological processes have been a significant bottleneck to rapid and efficient analysis of 3D cell culture models.…”