3D cell culture is a method for growing biological cells in an environment that allows them to expand and interact in three dimensions. This contrasts with older 2D techniques that confine cell growth to a flat surface. These cultures more accurately mimic the natural conditions found inside a living organism, facilitating the study of complex cellular behaviors not observable in two-dimensional systems. 3D culture systems were developed to create more physiologically relevant models for biological research.
From Flat Layers to Complex Structures
For many years, 2D cell culture was the standard, where cells are grown on flat plastic surfaces like petri dishes to form a single flat layer called a monolayer. While this approach is simple and low-cost, its flat, rigid environment does not accurately represent the intricate architecture of living tissues.
In a living body, cells exist within a complex microenvironment, interacting with neighboring cells and a supportive structure known as the extracellular matrix (ECM). These interactions influence cellular shape, movement, and function. Cells in tissues also experience gradients of oxygen, nutrients, and signaling molecules, meaning not all cells have equal access to these resources. Traditional 2D cultures fail to replicate these conditions, as all cells in a monolayer have uniform access to the culture medium.
This disconnect between the 2D lab environment and the 3D reality of the body can lead to misleading results, particularly in drug response or disease progression. Cells grown in flat layers often exhibit different gene expression, morphology, and proliferation rates compared to their counterparts in living tissue. 3D cell culture was developed to address these shortcomings by providing a model that better simulates the physiological complexity of an in vivo system.
Techniques for Building 3D Cell Models
Two primary strategies exist for creating 3D cell cultures: scaffold-free and scaffold-based methods. Scaffold-free techniques rely on the natural tendency of cells to self-aggregate into three-dimensional structures. A common example is the spheroid, a dense, spherical cluster of cells. They can be generated by methods like the hanging drop technique, where droplets of cell suspension are hung from a culture dish lid, allowing gravity to encourage aggregation.
Another scaffold-free approach uses specialized plates with low-adhesion coatings that prevent cells from sticking to the plastic, encouraging them to aggregate. In these spheroids, the cells create their own microenvironments, including gradients of oxygen and nutrients from the outer to the inner layers. This structure is similar to that of a small tumor and more closely resembles conditions inside living tissue.
Scaffold-based techniques provide an external supportive structure for cells to grow on or within. These scaffolds are made from porous biomaterials, such as hydrogels or polymeric fibers, that mimic the body’s natural extracellular matrix. Hydrogels are water-swollen polymer networks, popular as their composition and stiffness can be tuned to resemble specific tissues. Cells are mixed into the hydrogel solution before it solidifies, trapping them in a 3D matrix to grow and interact.
Advanced scaffold-based models include organoids and bioprinting. Organoids are complex, self-organizing structures derived from stem cells that differentiate and assemble into miniature versions of organs, complete with tissue-specific cell types. Bioprinting uses 3D printing technology to precisely deposit layers of cells and scaffold materials, or “bio-inks,” to construct tissue-like structures with precise control over the design.
Applications in Disease Modeling and Drug Discovery
The ability of 3D cell cultures to better replicate human tissue has led to advancements in medical research. In cancer research, 3D tumor spheroids are widely used because they mimic the structure, cell-to-cell interactions, and drug resistance of actual tumors. The layered structure of a spheroid creates barriers to drug penetration, similar to a real tumor, allowing researchers to screen anti-cancer drugs and identify compounds more likely to succeed in clinical trials.
The development of new pharmaceuticals also benefits from 3D culture systems. Models of specific organs, such as the liver or heart, can be created to test the toxicity and efficacy of drug candidates. For instance, 3D liver models more accurately predict drug-induced liver injury because the cells maintain their specialized functions for longer. This predictive power helps identify harmful compounds early in the development process, reducing costs and the reliance on animal testing, a shift encouraged by the FDA Modernization Act 2.0.
Beyond general drug testing, 3D culture technology enables personalized medicine. Cells from an individual patient, such as from a tumor biopsy, can be used to grow patient-specific organoids in the lab. These “avatars” can be used to test a panel of different drugs to determine which is most effective against that person’s specific disease. This tailored approach moves away from one-size-fits-all treatments toward therapies designed for an individual’s unique biology.