Traditional cell culture was developed on simple, nonporous two-dimensional (2D) surfaces, which facilitated the expansion of this vital technique across the life sciences. Because cells in vivo interact with their environment in three dimensions, 3D cell culture tools, reagents, and techniques have led to the creation of more predictive in vitro cell models for diverse applications and disciplines including cancer research, drug discovery, neuroscience, and regenerative medicine.
3D cell culture models may be generally classified into two principal categories based on method: 1) scaffold-based methods using hydrogels or structural scaffolds and 2) scaffold-free approaches using freely floating cell aggregates, typically referred to as spheroids. The choice of method depends principally on the nature of the cells themselves, but also on the goals and purpose of the 3D culture.
In scaffold-based culture, cells are supported in all dimensions either by an artificial structure or by a polymer network known as a hydrogel. These hydrophilic networks may contain over 90% water, and can be composed of animal-derived extracellular matrix (ECM) proteins, or are available as animal-free synthetic formulations. Cells are embedded in hydrogels to simulate the in vivo extracellular matrix.
So-called ‘hard’ scaffolds may also be created using specialized cultureware with fibrous or spongelike structures, often composed of biodegradable materials like polycaprolactone or optically-transparent polystyrene to optimize imaging. Though these manufactured supports are less like the in vivo ECM, they may enhance reproducibility and facilitate cell retrieval from the culture.
When cells are not grown on supports, they may form 3D aggregates called spheroids, which secrete their own ECM to become more like native solid tissues. Common examples include cancer tumorspheres, which enable the study of oxygen gradients and nutrient access in tumor formation. Spheroid culture is often favored for high-throughput compound screening in drug development and toxicology, where spheroids present more biologically-relevant models than 2D cultures. Spheroid culture can be achieved in diverse environments including low-attachment microplates, bioreactors, and microfluidics culture systems. Both scaffolded and scaffold-free systems enable interaction in all directions with substrate, other cells, and extracellular factors.
Advanced 3D cell systems allow researchers a hybrid between the accessibility of classical 2D cell culture techniques and the biological relevance of in vivo animal models, with fewer ethical concerns. Recently, the use of advanced 3D cell culture methods such as tumor spheroids, stem cell- and patient-derived organoids, and tissue engineering via 3D bioprinting with cells and bioinks have been implemented to more closely model in vivo cellular responses. Organoids derived from iPS cells have become commercially available for selected tissues, enhancing the potential for reproducibility and accelerating results versus lab-cultured organoids.
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