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Microbiology JoVE (Open Access) Citable · DOI

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

DOI: 10.3791/3669-v
What you'll learn
  • ✓ Perform in situ liver perfusion and collagenase digestion on rat tissue
  • ✓ Isolate pure portal fibroblasts without culture passage
  • ✓ Apply size selection to enrich fibroblast populations from liver cell suspensions
Protocol

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Difficulty
advanced
Total time
~2–3 hours per rat liver
Model organism
Rat (specific strain not stated)
Biosafety
BSL-1

Steps

1
Perfuse rat liver with collagenase in situ

Establish vascular access and perform in situ liver perfusion with collagenase solution to enzymatically digest the tissue while maintaining organ integrity.

▶ 01:20
2
Prepare biliary tree and digest liver tissue

Remove the liver and perform ex vivo digestion of the liver slurry after biliary tree preparation to further dissociate tissue and release fibroblasts.

▶ 03:31
3
Select portal fibroblasts by cell size

Apply size-based selection methods to isolate a pure population of portal fibroblasts from the digested cell suspension without requiring culture passage.

▶ 03:31
4
Characterize isolated primary portal fibroblasts

Verify the identity and purity of the isolated portal fibroblast population using representative microscopy or morphological assessment.

▶ 06:42
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