The need for new tools and technologies to decrease the cost of drug discovery and reduce animal testing has been long discussed. Recently, the director of the Center for Drug Evaluation and Research at the FDA called for “new evaluative tools for predicting, understanding, and assessing the effects of [drugs] in the relevant species (people).” Such tools have potential for reducing costly late-stage failures by providing an in vivo-like early-stage evaluation of efficacy and toxicity.
Three-dimensional (3D) cell cultures, in formats such as spheroids, gels, scaffolds, and bioreactors, have great potential as predictive tools in drug discovery. 3D cell cultures possess many features that mimic the in vivo microenvironment, which are lacking in traditional two-dimensional (2D) cell cultures such as physiological cell-cell and cell-extracellular matrix (ECM) interactions and mass-transfer gradients.
With the implementation of 3D cell cultures into the drug screening process, more physiological data can be obtained long before animal testing. Companies can then make better-informed decisions about which compounds are most promising, lowering the cost and time to get a drug to market.
Cellular spheroids, self-assembled microscale 3D aggregates of cells, have significant potential as models for drug screening. They are reproducible, versatile, and well-characterized. Spheroids possess physiological cell-cell contacts, secrete their own ECM, have nutrient, drug, and oxygen mass transfer gradients, and are often used as 3D models of many types of avascular tissues, tumors, and embryoid bodies.
3D Biomatrix Perfecta3D® Hanging Drop Plates facilitate easy and consistent spheroid formation in a 96- or 384-well format. Users pipet a cell suspension into each well, and, due to the well geometry, the suspension hangs below it (Figure 1). Since there is no surface to attach to, the cells aggregate and form a spheroid in the well.
The hanging drop confines spheroid formation to one per well, allowing the user to control the spheroid diameter with the type and number of cells in each well. Spheroids composed of 50–15,000 cells have been formed in Perfecta3D Hanging Drop Plates. Access holes allow for media exchange and the addition of compounds, reagents, or additional cells.
This article presents data on utilizing spheroid cultures to evaluate anticancer drugs and to build tissue and tumor co-culture models. This data demonstrates the potential of spheroid cultures as a predictive drug discovery tool.
Testing Drug Mechanisms and Efficacy
In a published study, the efficacy of two anticancer drugs with distinctly different mechanisms was compared in 3D spheroid cultures and traditional 2D cultures. 5-fluorouracil (5-FU) is a compound that inhibits cellular proliferation, and tirapazamine (TPZ) is a hypoxia-trigger cytotoxin that causes DNA damage.
Both 3D spheroid and 2D cultures of A431.H9 human epithelial carcinoma cells were treated with 10 µM of either 5-FU or TPZ. The data in Figure 2 show that A431.H9 cells are more resistant to 5-FU under 3D spheroid than 2D culture. However, the opposite is true for treatment with TPZ. A431.H9 cells were more resistant to TPZ when cultured in 2D than 3D spheroid culture conditions. A combination drug treatment of 5-FU and TPZ on the 3D spheroids demonstrated an additive effect (partial data shown in Figure 2 inset).
The discrepancy between the 2D and 3D data stems from the mechanism of each drug and its efficacy in each cell culture configuration. 5-FU targets proliferating cells. Therefore, it is more effective against the proliferating cells that comprise the 2D monolayer culture and suggests that cells have varying proliferation rates, including some quiescence, in the spheroids.
In contrast, TPZ is a hypoxia-activated cytotoxin. It is more effective in the spheroids, where both oxygen consumption and diffusion gradients create a hypoxic core similar to that of solid tumors. The additive effect of the two drugs suggests that the 5-FU killed the proliferating cells on the outside of the spheroid, and TPZ targeted the cells at the hypoxic core.
These data exemplify how 3D spheroid cultures can reveal efficacy data that is not always evident in 2D cultures. For thorough evaluation of compounds, 3D spheroid cultures offer an environment that mimics avascular tissues and tumors.
Building More Realistic Models
3D cultures can be made more relevant by including two or more cell types representative of the tissue or disease being studied. One cell type cultured in isolation may respond dramatically different to a compound than it would in vivo where multicellular interactions are influential. Co-cultures also can be used to study the effects of cell-cell interactions on cellular migration, metastasis, and differentiation.
The ability to access spheroid cultures from the top of the Perfecta3D Hanging Drop Plates gives the user access to several configurations of co-cultures (Figure 3). Homogeneously mixed co-cultures were obtained by mixing several cell types prior to seeding in the plates. Concentric spheroids were obtained by seeding one cell type, allowing time for spheroid formation, and then adding a second cell type.
Janus spheroids were achieved by separately culturing spheroids of different cell types before pipetting one spheroid directly onto another through the top of the well, where the two spheroids then merge together.
In each co-culture type, the ratio of cell types can be altered to best represent the tissue or disease being modeled; for example, a representative ratio of cancerous to noncancerous cells can be used to study metastasis and the effect of microenvironment on cancer growth.
The flexibility of cellular configuration and number make spheroid co-cultures particularly useful for studying the effects of a drug on the interactions between several cell types in cancer or stem cell research.
Spheroid models have great potential as drug-evaluation tools as they exhibit in vivo-like cell-cell and cell-ECM interactions, possess metabolic, proliferative, and diffusion gradients, and are simple to use on a large scale. The introduction of multiple cell types into 3D spheroid models further increases the relevance of the models. 3D cell cultures can be beneficial early on in drug screening, especially when evaluating compounds with mechanisms dependent on proliferative rates or oxygen concentration.