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 &#8…

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

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記事を引用
Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model. J. Vis. Exp. (Pending Publication), e20343, doi: (2023).

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