Three-dimensional (3D) cell culture technology can better simulate the natural environment in which cells survive in organisms, and its natural conditions can maintain cell-cell interactions and more realistic biochemical and physiological reactions. In a 3D environment, cells respond more closely to them in vivo responses to endogenous and exogenous stimuli such as changes in temperature, pH, nutrient uptake, transport, and differentiation. Advantages of using 3D cell culture models in drug candidate evaluation compared to two-dimensional (2D) cell culture models include:
i) Integrate/increase cell-cell and cell-matrix interactions.
ii) The influence of site-specific stromal cells in the tumor microenvironment can be assessed.
iii) Different cell proliferation zones.
iv) Create oxygen, nutrient, metabolite, and catabolite gradients to simulate a solid tumor environment.
v) Uneven distribution of drug candidates results in uneven exposure of cells to test molecules.
3D cell culture is a technology for growing cells into 3D structures. A variety of 3D cell culture systems have been developed, driven by the desire of biochemical and biomedical scientists to better represent in vivo experimental systems. The application of 3D cell culture allows researchers to better understand tissue and cancer behavior. Currently available 3D cell culture technologies can be divided into two categories, scaffold-based and scaffold-free 3D cell culture.
Scaffold-Based 3D Cell Culture
For scaffold-based 3D cell culture, prefabricated scaffolds or matrices are used to mimic the extracellular matrix (ECM) in vivo. Once introduced, cells attach, migrate, proliferate and fill the gaps within the scaffold to form 3D structures. Scaffolds include a variety of materials with varying porosity, permeability, and mechanical properties. Scaffolds and matrices can be of natural/biological origin or by chemical synthesis.
Many common biomolecules are good candidates for 3D matrices/scaffolds, including fibronectin, collagen, laminin, and gelatin. In addition to providing attachment sites and support for cells, natural/biological scaffolds can provide the correct microenvironment for growth factors, hormones, and other molecules. These small molecules interact with cells and play key roles in gene expression and protein production. Scaffolds derived from natural resources can also promote proper cell behavior and function, resulting in increased cell viability, proliferation, and differentiation.
A variety of synthetic materials can also be used as 3D matrices/scaffolds, including polymers, ceramics, glasses, carbon nanotubes, nanofibers, and more. During the design and fabrication of scaffolds, a range of properties should be considered, including biocompatibility, wettability, mechanical properties and structure, and surface chemistry. The high degree of controllability and flexibility of synthetic scaffolds allows for a wide range of applications. By providing a surface for cell growth and incorporating essential nutrients and growth factors, they can easily confer and alter 3D cell growth with minor changes to cell culture procedures.
Scaffold-Free 3D Cell Culture
For scaffold-free 3D cell culture, various techniques can be applied to achieve three-dimensional cell culture. One of the most widely used strategies is the hanging drop plate (HDP). In the absence of a surface for attachment, cells self-assemble into 3D spherical structures. HDP takes advantage of this fact by utilizing hole-bottomed wells with an open top, which is different from traditional culture plates. The aperture size of the HDP bottom holes is carefully designed to be large enough to form discrete medium droplets suitable for cell aggregation, yet small enough to prevent droplet detachment during handling by utilizing surface tension. The formation of the final spherical structure takes several hours to days, and its size is controlled by the initial cell quantity seeded into the droplets.
Another plate suitable for creating tumor spheroids or multicellular tissue models is the ultra-low attachment (ULA) coated microwell plate, which minimizes cell adhesion and allows for spheroid formation. The circular, conical, or V-shaped bottoms of ULA-coated plates ensure the generation of consistent individual spheroids and facilitate positioning the spheroids in the center of each well. Additionally, 3D cell culture structures can be formed in microfluidic systems where perfusion flow is introduced into the culture environment. Microfluidic systems can continuously supply nutrients and oxygen while ensuring timely waste removal. With the introduction of perfusion flow and the combination of various physical and non-physical barriers, in vitro models can mimic in vivo conditions.
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