Abstract
Three-dimensional (3D) in vitro microphysiological cultures, such as spheroids and organoids, promise increased patient relevance and therapeutic predictivity compared with reductionist cell monolayers. However, high-throughput characterization techniques for 3D models are currently limited to simplistic live/dead assays. By sectioning and staining in vitro microtissues, researchers can examine their structure; detect DNA, RNA, and protein targets; and visualize them at the level of single cells. The morphological examination and immunochemistry staining for in vitro cultures has historically been done in a laborious manner involving testing one set of cultures at a time. We have developed a technology to rapidly screen spheroid phenotype and protein expression by arranging 66 spheroids in a gel array for paraffin embedding, sectioning, and immunohistochemsitry. The process is quick, mostly automatable, and uses 11 times less reagents than conventional techniques. Here we showcase the capabilities of the technique in an array made up of 11 different cell lines stained in conventional hematoxylin and eosin (H&E) staining, as well as immunohistochemistry staining for estrogen (ER), progesterone (PR), and human epidermal growth factor (Her-2) receptors, and TP53. This new methodology can be used in optimizing stem cell-based models of disease and development, for tissue engineering, safety screening, and efficacy screens in cancer research.