Associated Challenges of Immunostaining Cells Encapsulated in Hydrogel Scaffolds

As 3D cell-based models continue to grow in popularity it is natural to try and apply existing 2D assays as a way of expanding experimental capabilities, but it is important to remember that large changes in model format can result in new experimental challenges that must first be overcome before useful data can be generated.

A common request that we often get at Visikol is if we can immunolabel microtissues encapsulated or grown in Matrigel; one of the first 3D cell culture options commercially available and a well know protein-based scaffold. Using Matrigel can result in cells expressing exciting biological phenotypes, but it can also introduce some unexpected complications with antibody labels. When immunolabeling tissues and microtissues the challenge is to get even labeling from the primary antibody. When you encapsulate tissues inside a hydrogel you have introduced an additional physical obstacle that the antibodies must first move through before they can begin to diffuse into the tissue. Typically, this can result in a high fluorescent background signal from the nonspecific interactions between the antibody and the scaffold, and makes the data processing more arduous or impossible. Using a hydrogel also can take solvent-based tissue clearing off the table as dehydration is an important step to prevent clouding due to the immiscibility of water with the clearing reagents. Since hydrogels rely on water to maintain their structure the dehydration will either succeeded and the hydrogel will be physically disrupted damaging your carefully constructed model or the dehydration will fail, and the clouding will make imaging impossible. Hydrogels can also complicate washing steps and liquid removal.

There are plenty of good reasons to grow microtissues in Matrigel, but there are also some bad ones. Don’t assume your cells require a scaffold-based system to aggregate, plenty of cells will assemble themselves into robust spheroids when seeded into low adhesion environments. Depending on your research question you might be able to save processing and analysis time by avoiding scaffolds. If Matrigel, or another scaffold, is required for your disease model, you may be limited to small molecule dyes. There are also synthetic scaffold alternatives to consider that may be more compatible with antibody labels.

Some examples of HepG2 spheroids grown and encapsulated in different scaffolds are presented below. The DAPI stain had the most success, but it does show some nonspecific labeling in the peptide scaffold. The Pan CK antibody label was very much impeded by the Matrigel and Alginate scaffolds resulting in high background fluorescence and unspecific labeling. This labeling could be improved by using a lower weight percent of hydrogel or by digesting the hydrogel before the labeling steps, but both options will have an effect on the physiology of the model. This is not to say never use an extracellular scaffold as a part of your cell model, but you should be aware of the potential labeling problems that it could introduce.

Blog Post By Dr. Peter Worthington

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