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2D and 3D Cell Culture Comparison

By , MD, PhD

Cell culture systems are indispensable tools that are used in a wide range of basic and clinical in vitro research studies. The classically preferred model is a static dish culture system which mainly generates adherent two-dimensional (2D) cell monolayers.

Still, such culture systems do not reflect the situation in vivo, where cells grow within a complex three-dimensional (3D) microenvironment and where vascular perfusion continuously supplies and removes metabolites and catabolites, respectively. Thus 3D cultures were introduced in order to improve the simulation of such conditions in the living organism.

2D vs 3D cultures

Today it is well known that cells behave structurally and functionally different when seeded on thin 2D surface-coated substrate versus a thick layer of polymeric 3D molecules, which more closely mimics their natural environment. 3D cultures exhibit a higher degree of structural complexity and homeostasis (i.e. the tendency to maintain a steady state), which is analogous to tissues and organs.

Main advantages of 2D cell cultures are easier environmental control, cell observation, measurement and eventual manipulation in comparison to 3D cultures. Furthermore, a rich body of literature exists to which outcome measures can be compared.

However, limitations such as inability to depict traits exhibited by in vivo systems (e.g. altered gene expression), decreased compatibility with in vivo systems, increased drug sensitivity and exposed surface hamper their use in both clinical and fundamental investigations.

Advantages of 3D cell cultures are proximity of other cells on all sides, behavior akin to in vivo conditions, as well as more accurate representation of cytoarchitecture. These types of cultures can also be used as efficient simulators of tumor characteristics such as dormancy, hypoxia and anti-apoptotic behavior.

Diffusional transport limitations for oxygen and other essential nutrients, as well as culture-dependent alterations in gene expression are potential drawbacks of the utilization of 3D cultures. Furthermore, some 3D cultures created from specific tissues (for example, basement membrane extracts) can sometimes contain undesirable components like viruses or growth factors.

It also must be noted that the increasing use of 3D culture techniques in different research areas is accompanied by technical challenges for microscopy. Whereas 2D cultures can be conveniently analyzed by almost any kind of imaging, 3D culture systems have to be optimized and specifically prepared for most experimental approaches.

Culture configurations

Most 2D cultures are adhesion dependent and cannot be grown in suspension cultures without mechanical support. All freshly isolated, immortalized and culture expanded cells have been cultured on tissue culture polystyrene plastic for many decades, where cells are spread to form focal adhesions.

Different types of 3D cultures can be made, including reaggregate or sphere cultures, hydrogel/scaffold cultures, rotary bioreactor cultures with cell aggregates or microcarriers, as well as organotypic slice cultures. These differ in terms of cell dispersion and preservation of tissue function.

Reaggregate cultures can be produced by rotation-induced reassociation or by providing conditions that promote sphere formation. Despite certain impediments in controlling extracellular components and the cellular distribution of reaggregate cultures, these can serve as great models to study cell-cell interactions.

Organotypic cultures are basically slices of tissue with maintained architecture, therefore preserving networks in the cut plane. This is different in comparison to all the other mentioned 3D culture types which generally utilize dissociated cells which reorganize in accordance to cell type, their adhesiveness and media conditions.

In conclusion, fundamental biological questions have been and will continue to be solved using 2D culture models. Nevertheless, 3D alternatives are pivotal in overcoming 2D matrix interactions that can fundamentally change cell behavior. In addition, the creation of large tissue-engineered constructs will necessitate a means of 3D fabrication with precise spatial arrangement of the contents.

Sources

  1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026212/
  2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386065/
  3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352326/
  4. http://onlinelibrary.wiley.com/doi/10.1002/jcp.24683/pdf
  5. LaPlaca MC, Vernekar VN, Shoemaker JT, Cullen DK. Three-Dimensional Neuronal Cultures. In: Berthiaume F, Morgan JR, editors. Methods in Bioengineering: 3D Tissue Engineering. Artech House, Norwood, 2010; pp. 187-204.
  6. Hess MW, Pfaller K, Ebner HL, Beer B, Hekl D, Seppi T. 3D Versus 2D Cell Culture: Implications for Electrom Microscopy. In: Müller-Reichert T. Electron Microscopy of Model Systems. Academic Press (Elsevier), 2010; pp. 649-671.

Last Updated: Jan 13, 2016

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