Cell culturing in traditional 2D systems has been around since the mid - late 20th century, and since then there has been a need for techniques to view and image them. Because of the flat, thin shapes cells adapt one of the most common methods for viewing live cells in 2D systems is phase contrast microscopy. However, this technique is typically not appropriate for 3D growth as cells adopt a more spherical shape in these systems. Additionally, the fibers that make up the scaffolds often cloud the outline of the cells. Complex three dimensional systems consequently require other tools to be visualized effectively.
Cellevate 3D Cell Culture plates are engineered to have a thickness that provides cells with a true 3-dimensional micro environment while maintaining a certain translucence, thus allowing for easy and good quality imaging and analysis. Our products are compatible with standard analytical instruments and visualization techniques. Below follows a brief summary of the most common types of microscopy used for imaging cells within 3D cultures.
Fluorescence microscopy is one of the most common ways to image cell cultures. For a sample to be suitable for fluorescence microscopy it must be fluorescent. There are several methods of creating a fluorescent sample; the main techniques are labelling with fluorescent stains or, in the case of biological samples, expression of a fluorescent protein. Cellular components can be stained using standard techniques and protocols (more information can be found in our “Protocols” section).
Confocal microscopy is a great analytical instrument to obtain high-resolution images of cells grown in 3-Dimensional scaffolds and to, for example, investigate and compare cell migration throughout the scaffold. As with fluorescence microscopy, samples must be fluorescent in order to be seen.
Electron microscopy can be used to obtain high-magnification images and can give good visualization of cell-to-cell and cell-to-fiber interactions. Cells cultured in scaffolds needs to be prepared in the same way as biological sample preparation. To maintain the cell fixation and the scaffolds structural shape, we recommend gradual dehydration using increasing concentrations of Hexamethyldisilazane (HMDS) or ethanol incubation. Recommended instructions for this can be found under “Protocols”.
Live imaging of cells in 3D scaffolds is difficult using conventional microscopy, due to the very limited focal depth of most microscopes. This makes it difficult to obtain sharp, focused images of cells, as they move out of focus when they migrate around in the scaffold. A good alternative for live imaging of cells in 3D is phase holographic microscopy, where the focus related problems have been solved using digital autofocus through computer algorithms, allowing for sharp images of cells regardless of where they are located in the scaffold matrix.