Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology

• Published in: Biomedicines 2022-09, Vol.10 (9), p.2198
• Main Authors: Poggel, Carsten, Adams, Timo, Janzen, Ronald, Hofmann, Alexander, Hardt, Olaf, Roeb, Elke, Schröder, Sarah K., Tag, Carmen G., Roderfeld, Martin, Weiskirchen, Ralf
• Format: Article
• Language: English
• Subjects: Animals; automated isolation; Automation; Calcium buffering; Cannulation; cell culture; cell separation; Centrifugation; Collagen; Collagenase; Communication; Enzymes; Experiments; Extracellular matrix; Health aspects; hepatocyte; Hepatocytes; Liver; Liver cells; Medical research; Metabolism; Perfusion; primary liver cells; Proteins; Reproducibility
• ISSN: 2227-9059; 2227-9059
• DOI: 10.3390/biomedicines10092198

Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes.