Home › Microbiology › Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol
Microbiology JoVE (Open Access) Citable · DOI

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

DOI: 10.3791/57400-v
What you'll learn
  • ✓ Isolate pure, viable endothelial cells from human colorectal tissue using enzymatic digestion and FACS
  • ✓ Perform flow cytometry-based sorting and characterization of tumor versus normal endothelial cells
  • ✓ Apply endothelial cell isolation for downstream drug testing and disease pathogenesis research
Protocol

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Difficulty
advanced
Total time
~4–6 hours per sample (tissue procurement through cell harvest and characterization)
Model organism
Human colorectal tissue (normal colon and colorectal carcinoma)
Biosafety
BSL-2

Steps

1
Prepare tissue and generate single-cell suspension

Surgically obtain human colon and colorectal carcinoma samples, mince tissue, and enzymatically digest to generate a single-cell suspension suitable for downstream sorting.

▶ 00:51
2
Perform FACS sorting and harvest endothelial cells

Apply fluorescence-activated cell sorting (FACS) to isolate endothelial cells from the suspension, then harvest and prepare sorted cells for characterization and culture.

▶ 04:17
3
Characterize endothelial cells and validate markers

Analyze isolated endothelial cells for expression of endothelial-specific markers (e.g., CD31, VE-cadherin) to confirm purity and distinguish tumor from normal endothelial populations.

▶ 07:17
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