Home › Cell Biology › In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion
Cell Biology JoVE (Open Access) Citable · DOI

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

DOI: 10.3791/3138-v
What you'll learn
  • ✓ Isolate primary liver sinusoidal endothelial cells (SECs) from rat liver tissue
  • ✓ Perform liver perfusion and differential centrifugation for cell purification
  • ✓ Track ligand internalization in hepatocytes and SECs using labeled compounds
  • ✓ Culture and prepare primary SECs for downstream experimentation
Protocol

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

Difficulty
advanced
Total time
~3–4 hours per rat liver (including perfusion, digestion, and differential centrifugation)
Model organism
Rat (Sprague-Dawley or Lewis)
Biosafety
BSL-1

Steps

1
Purify hepatocytes and SECs from rat liver

Perform liver perfusion and enzymatic digestion to isolate mixed liver cell populations. Use differential centrifugation to separate hepatocytes from sinusoidal endothelial cells based on sedimentation rates.

▶ 02:37
2
Incubate cells with labeled ligand internalization marker

Expose isolated hepatocytes and SECs to radioiodinated ligand (125I-SA-b-Hep) or similar labeled compound to monitor endocytosis. Measure uptake kinetics to assess cell-specific receptor-mediated internalization.

▶ 04:18
3
Purify hepatocytes and sinusoidal endothelial cells

Complete sequential centrifugation steps to obtain purified SEC and hepatocyte fractions. Verify cell purity and viability using standard microscopy and vitality assays before culture or experimentation.

▶ 06:55
4
Visualize biotin-labeled heparin distribution patterns

Apply fluorescent or enzyme-linked biotin-heparin to purified cells to determine tissue-specific binding and uptake distribution. Document localization data to confirm SEC-specific endocytic activity.

▶ 11:08
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