Cell Biology JoVE (Open Access) Citable · DOI

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

Name: Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy
Uploaded: 2026-06-04
Duration: 6 min 33 s
Description: The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

DOI: 10.3791/52483-v

What you'll learn

✓ Isolate crypts from human small intestinal and colonic tissue samples
✓ Culture enteroids and colonoids in basement membrane matrix
✓ Maintain and passage human epithelial organoids from surgical and biopsy specimens

Protocol

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Difficulty

intermediate

Total time

~5–7 days per sample (including initial isolation and first passage)

Model organism

Human intestinal epithelial tissue

Biosafety

BSL-1

Steps

Isolate crypts from whole tissue

Mechanically and enzymatically dissociate surgical intestinal tissue to release intact crypts for culture initiation.

▶ 01:14

Culture crypts in basement membrane matrix

Embed isolated crypts in Matrigel or equivalent basement membrane extract and establish 3D organoid culture conditions.

▶ 03:31

Evaluate enteroid and colonoid establishment

Monitor crypt outgrowth and organoid morphology from whole tissue and biopsy-derived samples to confirm successful culture initiation.

▶ 04:27

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