An optimized method for mouse liver sinusoidal endothelial cell isolation

• Published in: Experimental cell research Vol. 349; no. 2; pp. 291 - 301
• Main Authors: Meyer, Jeremy, Lacotte, Stéphanie, Morel, Philippe, Gonelle-Gispert, Carmen, Bühler, Léo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.12.2016
• Subjects: 60 APPLIED LIFE SCIENCES; Animals; BIOLOGICAL FUNCTIONS; CD11b; Cell Separation - methods; DIGESTION; DISPERSIONS; ELECTRON SCANNING; Endothelial Cells - cytology; Endothelium - cytology; FLUORESCENCE; Hepatocytes - cytology; IMPURITIES; LIPOPROTEINS; LIVER; Liver - cytology; LIVER CELLS; LSEC; MACROPHAGES; Male; MICE; Mice, Inbred C57BL; Microscopy, Electron, Scanning - methods; PURIFICATION; SORTING; Stabilin-2; SSC; HDMS; Purification; LSEC; MACS; FACS; FT; CD11b; P; FCS; Stabilin-2; Ac-LDL; Mice; SN
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2016.10.024

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. •This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions.•The liver cell dispersion step was improved by performing a retrograde cannulation of the liver.•The cell yield and the purity obtained were higher than comparative techniques in mice.•Contaminating macrophages were removed by introducing a CD11b- magnetic –activated cell sorting step.