Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model

- 3D organotypic models of colorectal cancer are in-vitro cultures engineered to mimic in-vivo tumor microenvironments. The culture often contains stromal cells, like fibroblasts, co-cultured with colorectal cancer epithelial cells, in a suitable extracellular matrix microenvironment.

In established cultures, tumor cells with metastatic potential invade the extracellular matrix. To isolate these invading cells, first, treat the organotypic model with formaldehyde to fix cells and extracellular matrix. Incubate with alcohol to dehydrate the sample.

Next, embed in liquid paraffin wax to generate a tissue block. Use an ultramicrotome to obtain thinly sliced sections. Mount the sections onto an appropriate glass slide.

Add Xylene to deparaffinize the sections. Sequentially, incubate the sections in decreasing concentrations of ethanol to rehydrate samples.

Treat with Cresyl Violet to stain and differentiate colorectal cancer cells from the microenvironment. Place the stained glass slide face down on the microscope stage. Using appropriate software, identify the cells to be microdissected and delineate the borders. Fire the laser to cut the highlighted section and collect the microdisse