An optimized method for mouse liver sinusoidal endothelial cell isolation
An optimized method for mouse liver sinusoidal endothelial cell isolation
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DOI：
10.1016/j.yexcr.2016.10.024
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发表时间：
2016-12-10
影响因子：
3.7
通讯作者：
Buhler, Leo
中科院分区：
文献类型：
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作者：
Meyer, Jeremy;Lacotte, Stephanie;Buhler, Leo
本研究的目的是建立一种准确、重复性好的小鼠肝窦内皮细胞分离方法。采用改良的胶原酶两步消化法结合Optiprep密度梯度离心法分离非实质细胞。用短期选择性贴壁法和CD146+磁激活细胞分选法(MACS)进一步纯化LSEC，并与我们的CD11b细胞去除和长期选择性贴壁相结合的纯化技术在细胞产量、活性和纯度方面进行比较。使用我们的技术纯化的LSEC每肝可获得7.07+/-380万LSEC，而CD146+MACs和短期选择性贴壁分别获得2.94+/-1.28和0.99+/-66万LSEC。用本方法制备的细胞纯度可达95.10+/-2.58%。CD146+MACs和短期选择性贴壁的纯度分别为86.75+/-3.26%和47.95+/-9.82%。同样，使用我们的技术进行纯化时，非LSEC的污染是最低的，污染巨噬细胞的比例仅为1.87+/-0.77%。此外，通过扫描电子显微镜分析的分离细胞呈现出典型的以筛板组织的LSEC窗口，证明该技术允许分离真正的LSEC。总之，我们描述了一种可靠和可重复性的技术，用于在小鼠体内分离高产率的LSEC。该方法为研究LSEC的生物学功能提供了一种有效的制备方法。
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.