Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model
Published: April 30, 2023
Abstract
Source: Bullock M. et al., Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts. J. Vis. Exp. (2014).
The video describes the laser dissection of the 3D organotypic co-culture model with micron-level precision. In 3D organotypic models, stromal constituents such as fibroblasts are 3-dimensionally co-cultured with cancer epithelial cells. The 3D model serves as an experimental tool that enables invasion and cancer-stroma interactions to be studied in near-physiological conditions.
Protocol
1. Organotypic Preparation: Preparation of the fibroblast impregnated organotypic gel: Prepare a fibroblast cell suspension containing 5 x 105 cells per organotypic in DMEM supplemented with 10% FCS and 292 μg/ml L-Glutamine. Make up the organotypic gels on ice in the following ratios: • 7 volumes of Rat-Tail Collagen: Matrigel mix (1:1) • 1 volume of filtered 10x DMEM …
開示
The authors have nothing to disclose.
Materials
| Colorectal cancer cell lines – example shown SW480 | ATCC | ATCC CCL-228 | |
| Collagen | BD Biosciences | 354265 | |
| Matrigel | BD Biosciences | 354234 | |
| Nylon membrane | Merck Millipore | VVLP01300 | |
| Metal grid | The Mesh Company | WSS20-A4 | themeshcompany.com |
| Laser microdissection platform | Leica Microsystems | Leica AS LMD | |
| Membrane mounted slides | Molecular devices | ||
| Cresyl Violet | Merck Millipore | 1052350025 | |
