Choosing the Right Substrate

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Many primary cells have difficulty adhering to uncoated glass and plastic surfaces, especially in low serum or serum-free conditions. For primary cells, it is necessary to use a substrate coating to enhance cell attachment. There are many different substrates to choose from and each type can have dramatically different effects on cell attachment, morphology, and proliferation. It is important to consider these factors when selecting a substrate to use for a particular cell type.

First, consider the difference between plastic and glass. Tissue culture-treated plastic is modified to be hydrophilic and substrates will coat the surface more thoroughly. Glass coverslips, depending on the manufacturer and how the glass was treated, can be hydrophobic or have residues from manufacturing that hinder substrate binding. Some researchers utilize pre-treatment protocols like acid washing, flaming, and/or autoclaving to enhance substrate binding, though this may not always be necessary. While numerous strategies exist to treat culture glass, some cell types will still have difficulty attaching on glass.

There are two main categories of substrates: naturally-occurring and synthetic. Natural substrates, such as extracellular matrix, are extracted and purified from tissues for use in cell culture. The most commonly used natural substrates are collagen, fibronectin, and laminin. Cells interact with these matrix components via cell surface receptors, such as integrins. Receptors bind to domains of collagen, fibronectin and laminin and trigger intracellular signaling pathways that facilitate adhesion complex formation, and can also trigger cell proliferation or differentiation. Synthetic substrates, on the other hand, are not derived from animal by-products and may be advantageous for certain applications. The most common synthetic substrate for 2-dimensional culture includes poly-lysine, a polymer of lysine containing a positively charged amino group. The positive charge non-specifically enhances cell attachment by attracting the negatively charged cell membrane.

To illustrate how substrate choice can affect cell growth and morphology, we cultured Human Coronary Artery Endothelial Cells on Poly-L-lysine versus fibronectin. After 3 days, the cells showed higher proliferation on the fibronectin compared to Poly-L-lysine. While fibronectin is better in this particular case, Poly-L-lysine may be preferred for other cell types.

Substrate blog

In conclusion, substrate choice can have an enormous impact on how primary cells attach and grow. Primary cells have limited expansion capacity, so it is important to use the best substrate in order to get the most out of your experiments. Before proceeding with costly studies, it is a good idea to seek recommendations from colleagues and publications or test substrates in your own lab first. Alternatively, if you have purchased primary cells from a manufacturer, consult the cell product sheet for detailed information.


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