3D Spheroid Model Formation and Analysis
HCT116 human colorectal carcinoma cells were used to generate spheroids by seeding 20,000 cells in 96-well HDPs and 5,000 cells in 384-well HDPs. Cells were maintained in hanging drops for three days to permit spheroid formation. The resulting spheroids were uniformly sized and displayed characteristic morphologies as observed by light microscopy (Figure 3A).
Spheroids were transferred into 384-well, black-sided, clear-bottomed, high-content imaging assay plates (Greiner) by the addition of supplementary media. Viable cells were stained with calcein AM according to the manufacturer’s instructions. Spheroid size analysis (Figure 3B) was performed by capturing images via confocal microscopy.
The resulting spheroids had an average diameter of 650 µm from 96-well HDPs and 350 µm from 384-well HDPs (Figure 3B), demonstrating excellent reproducibility of spheroid size relative to initial cell plating number.
To gauge the applicability of 3D spheroids as a tumor model, sectioning and immunohistochemistry were performed to further characterize the tumor-like morphologies (hematoxylin and eosin stain, H&E), proliferative zones (Ki67 antibody), and presence of hypoxic adducts (pimonidazole, PIMO) of 384-well tumor spheroids. Although the spheroids were less than 500 µm in diameter, there was evidence of hypoxic cores, and proliferation was detected throughout the spheroids, particularly along the outermost layer (Figure 3C).
The ability to quickly and easily create multiple 384-well plates of spheroids with consistent size and biologically relevant microtumor physiology allows for high-throughput screening in 3D. Based on these observations, HDP-derived spheroids constitute a robust 3D tumor model for high-throughput drug discovery, especially when employing the acumen high-content imager for quantifying spheroid formation.