An optimized method for mouse liver sinusoidal endothelial cell isolation

作者：Meyer Jeremy; Lacotte Stephanie; Morel Philippe; Gonelle Gispert Carmen; Buhler Leo

来源：Experimental Cell Research, 2016, 349(2): 291-301.

DOI：10.1016/j.yexcr.2016.10.024

• LSEC
• Mice
• Purification
• CD11b
• Stabilin-2

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.

• 出版日期2016-12-10