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Assay Tutorials : Sep 15, 2011 ( )
Supporting Cells with Scaffold Technology
Polystyrene Platform Designed to Facilitate Routine 3-D Cell Culture!--h2>
Cell-based in vitro assays are a key component of research into basic cellular mechanisms, disease modeling, compound screening, and safety assessment. Cultured mammalian cells are particularly important tools for providing predictions of drug activity, metabolism, and toxicity in vivo. Conventional cell culture methods, however, provide a growth environment that is far removed from what cells experience in real-life tissues.
In vivo, cells grow naturally in three dimensions (3-D) and are supported by a complex extracellular matrix that facilitates cell-cell communication via direct contact and through the secretion of paracrine factors. These features change dramatically when cells are grown in the laboratory. When forced to grow on a flat two-dimensional (2-D) substrate, cells adapt and radically change their shape, which, in turn, influences their internal cytoskeleton and can subsequently effect gene and protein expression and, ultimately, cell function.
In addition, almost 50% of the cell rests against the flat plastic substrate and a similar area is exposed to the culture medium above. The opportunity for cell-cell interaction is minimal and is significantly reduced when cells grow as monolayer cultures.
These disadvantages are widely recognized in the scientific literature, and technologies are under development to improve the environment in which cultured cells grow. Demands for improvement include the obvious need to enhance cell function but also to improve the predictive accuracy of in vitro assays as a means to reducing subsequent development costs, advancing basic research, developing more relevant human models of cell function, and adapting to changes in policy concerning the reduction of animals in research.
Published research has clearly demonstrated that culturing cells in 3-D radically enhances cell growth, differentiation, and function. Authentic 3-D cell cultures provide greater insight into how cells behave in the body in response to external challenges than is currently possible with existing 2-D culture technologies.
The development of scaffolds and their use in cell culture is a proven approach that provides the additional vertical axis or third dimension into which cells can grow. However, there is currently no scaffold platform technology that supports genuine 3-D cell culture for routine use alongside conventional cell culture methods.
Ideally, such scaffold technology should satisfy several criteria for it to be successfully adopted by the scientific community. The scaffold itself should have a uniform and consistent structure and be manufactured with tight reproducible tolerances such that batches of the material are equivalent. This is an important factor since cells respond to their environment and will behave differently if the material is changed.
The scaffold must be highly porous to enable cells to enter and migrate freely throughout the material. However, the internal dimensions of the material should not be such that cells are unable to bridge gaps, fill the space, and develop 3-D cellular structures. In a way, the scaffold merely acts as a catalyst to initiate the process and enable cells to build up on one another creating the natural 3-D structure of a tissue.
Reinnervate’s alvetex® is a highly porous polystyrene scaffold. It is composed of voids and interconnecting pores and has a consistent structure (Figure 1). The scaffold is engineered into a thin membrane to aid cell entry and exchange of materials by passive diffusion.
Polystyrene is inert and will not degrade during an experiment. This is advantageous since biodegradability may introduce variability and cause local release of degraded material that may influence cell function. In addition, the fabric of a polystyrene scaffold is consistent with the material used for the majority of conventional 2-D cultures and users need not be concerned how their cells react to the material itself.
The advantages of growing cells in 3-D culture over existing 2-D methods are self evident in the literature and on dedicated websites. However, it is also recognized that some investigators are reluctant to change and it is the pioneers who will first work with such new technologies.
It is essential, therefore, that any radical changes to cell culture practice first result in technology and methods that are easy to use routinely, are readily adaptable to existing analytical procedures, and are well exemplified. Multiwell plates and well inserts are considered industrial formats for 2-D culture and a scaffold must be compatible with these existing types of product.
Alvetex satisfies all these criteria and is available in the base of multiwell plates and a custom well insert (Figure 2). Like plasticware for conventional 2-D culture, alvetex comes sterilized, prepacked, and ready to use. It is a cost-effective solution for routine 3-D cell culture, which is readily transferred between laboratories and its use does not require the purchase of any specialized equipment.
There are significant differences between how 2-D and 3-D cell culture is practiced and such differences often follow common logic. For example, in 3-D culture there tend to be many more cells per unit volume of medium compared to the equivalent 2-D system. Therefore, the frequency of media changes needs to be increased. However, this might not be the preferred or most convenient option.
Increasing the volume of medium available to support 3-D cultures has been shown to be a simple and effective way of growing cells undisturbed for several days (Figure 3). Similarly, feeding 3-D cultures from above and below the scaffold using a custom well insert improves long-term maintenance of cells.
Educating users to think differently about how to adapt current practice to the growth of their favorite cell type in 3-D will be critical to the adoption of 3-D cell culture technology. Equally important will be the availability of easy-to-use technology that is flexible and versatile, and enables users to practice 3-D cell culture routinely alongside conventional 2-D culture methods.
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