Visualizing Cells on XanoMatrix™ Scaffolds

The ability to visualize individual cells in vitro either fixed or live specimens on clear plastic is unmatched. However, to study the same behavior in an in vivo-like, 3-dimensional environment presents some challenges. Etching of a 2D surface or thin coatings provide some benefit over flat plastic, but do not meet the requirements of replicating deep ECM tissue.

Scanning Electron Microscopy

Preparing the scaffold for electron microscopy is straight forward and very similar to protocols for biological sample preparation. The key factor for 3D samples is maintaining the structural shape of the scaffold and the cells by fixation and gradual dehydration using increasing concentrations of ethanol incubation. Refer to the protocol for detailed steps.

Vac-High PC-Std. 10kV x900 (48mm)
Vac-High PC-Std. 10kV x900 (48mm)
Vac-High PC-Std. 10kV x1700 (48mm)
Vac-High PC-Std. 10kV x1700 (48mm)

The SEM images above show two magnifications of XanoMatrixTM scaffolds which have been seeded with MDA-MB-231 cells (left images) or incubated in media with no cells (right images) prior to fixation and sample preparation. The cells that grow on the nanofibers (top, left) show a flat morphology creating obvious shadows while cells growing on the microfibers (bottom, left) show visible cellular structures. Optimization of SEM parameters can also allow for investigation of structures of cells growing on the nanofibers.

Fluoroescence Microscopy

Phase contrast microscopy, while the standard for viewing live cells in 2D growth, typically is not appropriate for 3D growth due to the non-flat shape of the cells in the matrix. Additionally, the fibers which create the matrix often clouds the outline of the cells. However, XanoMatrix scaffolds have been designed with a thickness that allows for routine optical inverted fluorescence microscopy. To view under live fluorescence, the cells must either produce GFP or RFP or be vital dyed with CellTracker (Invitrogen) or a similar cell tracking product.

fluoro2
3T3optical

NIH/3T3 cells producing stable GFP (above) are easily viewed in culture under 488nm fluorescence inverted microscopy.

While high resolution imaging under optical microscopy is not ideal for 3D tissue culture given the z-axis growth component, viewing cell populations or colonies combined with a non-destructive cell metabolism assay such as AlamarBlue or MTT will give the researcher a high amount of qualitative data without sacrificing the cells for analysis.