Home › Immunology › Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context
Immunology JoVE (Open Access) Citable · DOI

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

DOI: 10.3791/3089-v
What you'll learn
  • ✓ Prepare collagen type I matrix from rat tail tendon
  • ✓ Embed primary human fibroblasts in 3D collagen gel
  • ✓ Assess cell invasion and migration in stromal context
  • ✓ Apply organotypic assay to study cell behavior in tissue-mimetic environment
Protocol

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Difficulty
advanced
Total time
~3–4 weeks (including fibroblast culture establishment, collagen extraction, matrix preparation, and invasion assay duration)
Model organism
Primary human fibroblasts; rat (collagen source)
Biosafety
BSL-1

Steps

1
Establish fibroblast cultures from skin explants

Isolate and culture primary human fibroblasts from skin tissue explants for use as stromal component in the organotypic gel.

▶ 01:44
2
Prepare collagen type I from rat tails

Extract and purify collagen type I from rat tail tendons to serve as the primary extracellular matrix component.

▶ 02:57
3
Set up 3D matrix with embedded fibroblasts

Combine collagen I with primary fibroblasts and allow polymerization to create a tissue-mimetic stromal gel substrate.

▶ 04:55
4
Plate cells of interest on top of matrix

Seed test cells (e.g., cancer or invasive cell lines) onto the surface of the established collagen-fibroblast matrix.

▶ 06:48
5
Transfer matrix to grid for invasion assay

Move the seeded matrix to a specialized grid or culture insert to enable long-term monitoring of cell invasion over 0–21 days.

▶ 07:33
6
Fix and process matrix for analysis

Chemically fix the gel and embedded cells at designated timepoints to preserve morphology and enable microscopic evaluation.

▶ 08:38
7
Review applications and assay adaptations

Explore variant applications of the organotypic platform for different cell types and imaging modalities.

▶ 09:02
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