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Cell Biology JoVE (Open Access) Citable · DOI

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

DOI: 10.3791/56993-v
What you'll learn
  • ✓ Perform portal vein catheterization and liver perfusion with collagenase
  • ✓ Isolate viable hepatocytes via differential centrifugation
  • ✓ Purify sinusoidal endothelial cells from liver digest
  • ✓ Assess cell viability and yield using standard analytical methods
Protocol

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

Difficulty
advanced
Total time
~60–90 min per mouse (surgery + perfusion + cell isolation)
Model organism
Mouse
Biosafety
BSL-1

Steps

1
Place catheter in portal vein

Surgically expose the mouse liver and insert a catheter into the portal vein to enable controlled perfusion with collagenase solution.

▶ 00:45
2
Perfuse liver and isolate hepatocytes

Perfuse the liver with type IV collagenase solution via the portal vein, then perform differential centrifugation steps to separate and collect viable hepatocytes.

▶ 02:46
3
Purify sinusoidal endothelial cells

Apply further centrifugation and density-based separation techniques to the remaining liver digest to isolate sinusoidal endothelial cells from hepatocyte-depleted fractions.

▶ 04:39
4
Analyze cell purity and viability

Perform flow cytometry and microscopy analysis on purified hepatocyte and SEC fractions to confirm cell identity, purity, and viability metrics.

▶ 07:18
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