While two-dimensional (2D) cell cultures are still being used for basic research and drug discovery, screening, and testing, the use of 3D cell cultures is fast gaining popularity since it effectively addresses many of the limitations commonly encountered in 2D cell cultures. Specifically, three-dimensional (3D) cell cultures:
- Imitate the structures of cells and tissues in their natural environment. While cell morphology and functions are preserved in 3D cell cultures, these characteristics are inevitably altered upon transferring cells and tissues to 2D conditions.
- Preserve cell interactions and molecular mechanisms. This is not possible in 2D cell cultures, since there is no proper representation of the cell-to-cell and cell-to-extracellular environment interactions responsible for cellular functions (e.g. differentiation, proliferation, vitality, gene expression, responsiveness to stimuli, and drug metabolism).
- Provide conditions similar to in vivo environments. Since cells are arranged in a monolayer in 2D cultures, they have unlimited access to oxygen, nutrients, and metabolites in the medium, a total contrast to in vivo conditions. In their natural microenvironment, cells and tissues have variable access to these essential compounds.
Types of 3D Cell Cultures
While 2D cell cultures grow cells in a flat monolayer on a plate, 3D cell cultures can be grown with or without a scaffold.
Scaffold-based 3D Cell Cultures
Using the scaffold technique, cells can be grown on a variety of culturing tools including solid scaffolds, hydrogels (e.g. animal extracellular matrix extract, protein, peptide, polymer, and wood nanocellulose hydrogels), and other materials.
In this method, the cells are free to migrate among the fibers and attach to the scaffold where they eventually grow and divide. Thus, the system is compatible with commercially available functional tests as well as DNA/RNA and protein isolation kits and can be easily prepared for immunohistochemical analysis.
There are several advantages in growing 3D cultures in hydrogels. Specifically, it:
- Allows cells to retain their normal physiological shape
- Can be customized to simulate the cell’s natural environment (including the rigidity or stiffness of the tissue of origin)
- Doesn’t require any sophisticated materials, protocol, or devices
Scaffold-free 3D Cell Cultures
3D cells can likewise be cultured on different media such as non-adherent plates, hanging drop plates, micro-patterned surfaces, and rotating bioreactors. Additionally, 3D cell cultures can be created via magnetic levitation method or magnetic 3D bioprinting.
- Non-adherent plates. The hydrophilic polymer coating in low adhesion plates inhibits cells from sticking to the surface and promotes the clustering of cells which encourages the formation of extracellular matrix.
- Hanging drop plates. In this scaffold-free method, the cells are placed in a suspended drop of medium to promote cell aggregation and encourage the formation of spheroids at the bottom of the droplet.
- Micro-patterned surfaces. 3D cell cultures can also be cultivated in micro-patterned surfaces or microwells.
- Rotating bioreactors. Cells are grown in small cylindrical plastic chambers made from bioactive synthetic materials. These chambers are designed to provide a nutrient-rich micro-environment for the growing culture, an easy access to the cell spheroids, and 100% humidity to promote maximum cell growth and function.
- Magnetic levitation method (MLM). This refers to the process of growing tissue cultures and promoting cell-to-cell interactions by levitating magnetic nanoparticle-treated cells up to the air/liquid interface of a standard petri dish. This method is extremely scalable and is capable of culturing anywhere from 500 to millions of cells.
- Magnetic 3D bioprinting. In this process, the cells are incubated or tagged with magnetic nanoparticles and printed into specific 3D patterns (rings or dots) using magnetic fields.
Disadvantages and Limitations of 3D Cell Cultures
- It can be time-consuming. Aside from the time-consuming process of preparing the culture media (in the case of hydrogels), proteolytic degradation may also be required to separate individual cells from spheroid structures. This process can take several hours to a few days.
- It has low reproducibility. Many 3D methods have low efficiency and repeatability compared to 2D cell cultures.
- The results may depend on the method used. Choosing the wrong model can negatively influence the results of the experiment.
