Mammalian cells usually need to adhere to a surface to grow. In the laboratory, tissue flasks and roller bottles providing two-dimensional surfaces are commonly used for simplicity. For industrial-scale processes, microcarriers can be used to provide larger surface areas for cell attachment in suspension cultures, but this is limited by the difficulty in seeding the microcarriers with cells. As an alternative, hollow-fiber membranes, polymer gels and foams, and fibrous matrices are being used for growing cells. These systems have advantages because their three-dimensional structures mimic the in vivo cell environment, which is of critical importance to cellular function and their applications.

Three-dimensional cultures provide high surface areas on which cells can attach and arrange themselves. For a similar reactor volume, a three-dimensional culture can provide a surface area three orders of magnitude larger than the flat surface of a two-dimensional culture. In three dimensions, cells are not limited by contact inhibition and can reach a higher density closer to that found in tissues, provided nutrients can be efficiently transported. Recent research has revealed that cells behave very differently when they are grown in three dimensions. The third dimension provides another direction for the cell interactions, migration, and morphogenesis, which are all important in regulating cell cycle and tissue function.

Cells attach along the matrix fibers, form bridges spanning adjacent fibers, and aggregate in the void space, all depending on the matrix porosity and pore size, factors which affect cell proliferation, differentiation, gene expression, and tissue function. However, very little has been done to ‘quantify’ the three-dimensional spatial effects on tissue development. Now that developments in polymer microfabrication and soft lithography can produce tissue scaffolds with well-defined three-dimensional structures1, we can study the effect of structure on the organization of cells and their interactions, which could lead to the optimal design of tissue scaffolds.

Although current applications of three-dimensional cultures are mostly limited to tissue engineering, a three-dimensional perfusion culture system can be used to produce recombinant proteins more efficiently for therapeutic purposes. For example, the higher cell density in a three-dimensional cell culture can produce more monoclonal antibody at a higher titer2. It is not known, though, if protein glycosylation differs in three-dimensional cultures from those in two dimensions. Three-dimensional perfusion cultures can also mass-produce cells, such as pluripotent stem cells, without needing subculturing or passaging of the cells to maintain their activity and normal cellular function3. For example, cow luteal cells cultured in a three-dimensional bioreactor can maintain their normal function for a longer period than cells in two-dimensional T-flasks. The ability of cells to maintain their normal function is critical in the development of cell-based assays for drug screening.

Three-dimensional bioreactors are generally better than two-dimensional models in predicting drug treatment efficacy. Colon cancer cells from three-dimensional cultures showed up to a 180-fold increase in drug resistance compared with cells cultured in two dimensions. A 1000-fold decrease in the cytotoxicity of gemcitabine was found in multilayer cultures of colon and ovarian cancer cells, while two-dimensional monolayer cultures erroneously predicted gemcitabine to be an effective proliferation inhibitor. Bioreactor models using two-dimensional cultures for cytotoxicity analyses are inherently prone to error because of their lack of three-dimensional structural support for cell growth and tissue function. In addition, cell-based sensors using two-dimensional cultures are difficult to implement in online monitoring because of the low signal intensities that result from the small number of cells on two-dimensional surfaces. On the other hand, three-dimensional cultures of cells expressing green fluorescent protein have been developed for cell proliferation and cytotoxicity assays that can be used for high-throughput drug screening.

Although the benefits of three-dimensional scaffolds in cell culture and tissue engineering are clear, directing cell function using biomaterials is challenging. Nanostructured polymers offering tunable ‘surface’ properties similar to the natural extracellular matrix can be incorporated into three-dimensional scaffolds. The higher surface energy stemming from nanoscale surface roughness may positively affect cell adhesion, proliferation, and function. Therefore, three-dimensional scaffolds with nanoscale features offer great promise for enhancing the biological performance of cell cultures, but they are not yet fully exploited.

Further reading
[1] Y. Yang et al. Biomaterials, 26 (2005), p. 2585
[2] S.T. Yang et al.,in: J.J. Zhong (Ed.), Adv. Biochem. Eng./Biotechnol., 87 Springer, Berlin, Germany (2004), p. 61
[3] A. Ouyang, S.T. Yang, Stem Cells (2006) doi: 10.1634/stemcells.2006-0322


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DOI: 10.1016/S1369-7021(07)70030-5