HSCs were isolated from the mouse liver and prepared as previously described (9 (link),14 (link)). Briefly, the liver was perfused with collagenase IV (Sigma-Aldrich, St. Louis, MO) solution (1.0 mg/mL) via the portal vein. After 30 minutes of digestion at 37°C, the isolated cells were filtered through a nylon mesh (200 microns mesh size). The HSCs were enriched by Percoll density gradient centrifugation (Sigma-Aldrich; 1.130 relative density for 10 min at 39,000g) and cultured in RPMI 1640 complete medium supplemented with 20% fetal bovine serum for 14 to 21 days. The purity of HSCs exceeded 90% determined by desmin immunostaining.
Protocol detail
Isolation and Culture of Mouse Hepatic Stellate Cells
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Cells
Collagenase
Cultured medium
Density gradient centrifugation
Desmin
Digestion
Fetal bovine serum
Hscs
Liver
Mouse
Nylon
Percoll
Portal vein
Corresponding Organization : Cleveland Clinic
Other organizations : Cleveland Clinic Lerner College of Medicine
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Variable analysis
independent variables
- Collagenase IV concentration (1.0 mg/mL)
- Collagenase digestion time (30 minutes)
- Percoll density gradient centrifugation (1.130 relative density for 10 min at 39,000g)
- Culture duration (14 to 21 days)
- Purity of HSCs (determined by desmin immunostaining)
- Liver perfusion via the portal vein
- Cell filtration through a nylon mesh (200 microns mesh size)
- Culture medium (RPMI 1640 complete medium supplemented with 20% fetal bovine serum)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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