Designing 3D Cell-Based Assays

3D cell-based model systems—designed to approximate the physiological complexity of cellular environments—are meant to simulate in vivo physiology better than their flat monolayer counterparts. As such, they are important in drug screening and disease research. “Our tissues and organs are three-dimensional structures that have gradients of gases, waste, and nutrients associated with them,” says Hilary Sherman, Senior Applications Scientist at Corning Life Sciences. “These gradients can have an impact on organ function, disease progression, and the success of drug delivery so it makes sense that we would want our models to replicate those gradients whenever possible.” Here is a look at the advantages of 3D cell-based assays, and expert advice on their design.

The importance of complexity

The inherent complexity of 3-dimensional tissues is one of the attractions of 3D cell-based assays. This physiological complexity is especially important when studying specialized structures such as tumors. “Morphology and polarity of the cells are maintained and 3D models show a similarity to cells growing in vivo in terms of cellular topology, gene expression, signaling, and metabolism,” says Kyung-A (Katie) Song, Scientific Support Manager at Corning Life Sciences. “Two-dimensional cell culture systems often do not reflect the biological characterization or drug sensitivity to targeted therapy on patient tumors.”

Indeed, it is becoming clear that different areas within a 3D tissue have different roles within a tumor, perhaps reminiscent of tumor microenvironments observed in vivo. “Depending upon their diameter and density, spheroids or microtissue masses assemble and grow into zonal regions with different solute permeability, rate of metabolism, and proliferative capacity,” says Andrew Niles, Senior Research Scientist at Promega. “Chemotherapeutics are designed and developed to address and circumvent this complexity because ultimately clinicians don’t treat monolayer tumors growing on polystyrene surfaces.”

3D assay design

Given the varied methods available for growing cells in 3D cultures, including with or without scaffolding material such as hydrogels, there are numerous concerns to take into account when designing a 3D cell assay. “Scaffold-dependent methods can provide tunable protein matrices to mimic the properties of native tissue, [but] scaffold-free methods tend to support higher-throughput applications and can provide a more optimal environment for modeling the behavior of some cancerous tissue,” says Joe Clayton, Global Scientific Program Manager, Cell Analysis Division at Agilent Technologies. “The goal is to achieve more predictive models for downstream drug discovery efforts while relying less on animal models.”

Assay design is in part dependent upon the cell type. Cell types that thrive with fewer attachments—such as in spheroid, ultra-low attachment (ULA) environments—are often “well- suited for drug discovery screening efforts because they are simple and scalable, and produce uniform spheroids throughout the assay plate,” says Niles. “[Cells requiring scaffolds or extracellular matrix hydrogels in] more amorphous mass models may be perfectly acceptable for basic research or proof-of-concept, but the data must be more rigorously normalized by other assays between control groups.”

Given the greater complexity of 3D cultures, it is smart to choose an assay that has been specifically designed and optimized to perform well with 3D cultures whenever possible. “3D-validated versions of common assays to measure cell viability, proliferation, and live/dead cells have been developed for 3D cell models such as spheroids and organoids,” says Nick Asbrock, Global Product Manager for Stem and 3D Cell Culture at MilliporeSigma. “For example, we have developed a 3D cell culture tested and optimized Live-Dead Cell Viability Assay Kit that consists of Calcein-AM (stains live cells), Propidium Iodide (stains dead cells) and Hoechst 33342 (stains all cells) that can be used for flow cytometry, fluorescence microscopy and with fluorescence microplate readers.” Compared to a 2D assay kit for the same purpose, the 3D kit uses different concentrations of dyes and buffers, having been optimized and validated on both spheroid and organoid cell models.

Another challenge is choosing the right 3D assay system, keeping in mind that greater complexity can also mean additional work and cost. Sherman notes the importance of choosing an appropriate level of complexity for your research questions. “More complex models that require multiple cell types or biological hydrogels might not add to the quality of the data depending on what is being studied,” she says. “That means that sometimes a simpler model such as that achieved with a low-attachment coating might be a better solution.”

It’s also a good idea to be aware of what types of data analysis will be involved when developing 3D cell assays—and to eschew any unnecessary number-crunching. Consider the amount of data, for example, generated by confocal fluorescence imaging used in quantitative 3D assays. “While high magnification, multi z-stack image acquisition may provide great sample detail, the resulting image acquisition and processing time, and associated file size, can become burdensome,” says Clayton. “For many applications, a single image, or a small z-stack, focused on the centroid of the sample is sufficient to quantify relative differences between conditions.”

Assay limitations and challenges

Though 3D cell culture is becoming easier, some cell types are more challenging to culture in three dimensions—especially those with more complex growth requirements. “While many cell lines will readily form compact cell aggregates or spheroids, primary cells and stem cell populations often require optimized conditions, including matrix support and co-cultures to achieve desired results,” says Clayton.

An inherent limitation of 3D tissues is making the reagents accessible to all the cells equally. Indeed, this is one of the most consequential challenges of a 3D model system. “This challenge increases as a function of mass size and density, and can limit diffusion of cell permeable probes or adversely impact efficient cell lysis to extract analytes,” says Niles. “It is therefore important to understand the validated limits of a particular assay and in which model systems the reagents were tested.”

Similarly, 3D structures can also pose additional challenges to accessibility of antibodies when staining cells. “We have compiled a step-by-step protocol and list of organoid-qualified antibodies used to stain organoids for immunofluorescence microscopy using whole-mount immunostaining methods,” says Asbrock.

Imaging 3D structures is already more challenging, but additional obstacles can come from the often opaque animal-derived hydrogels used as extracellular matrix. “We have developed an alternative, chemically defined hydrogel for 3D cell culture that is optically clear and designed for microscopy,” says Vi Chu, Head of Cell Biology R&D at MilliporeSigma. “These hydrogels allow for higher-resolution microscopy to analyze complex structures seen throughout most 3D cell models.” Hydrogels and scaffolding may also limit reagent access, or interfere with light paths during imaging.

Variability in the data is always a concern for 3D cell-based assays. “Due to the complex nature of most 3D cell models, there can be a higher level of variability from well-to-well,” says Chu. “One method to reduce the variability is to use single-cell passaging when setting up experiments; another method is to manually screen from well-to-well and choose wells with similar sized 3D cultured cells before starting an assay.” Such practical solutions continue to evolve to support the success and advancement of 3D cell-based assays.