All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.
All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.
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DOI：
10.1371/journal.pone.0138655
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Broering R
中科院分区：
文献类型：
--
作者：
Werner M;Driftmann S;Kleinehr K;Kaiser GM;Mathé Z;Treckmann JW;Paul A;Skibbe K;Timm J;Canbay A;Gerken G;Schlaak JF;Broering R
肝细胞是先天免疫的关键参与者。因此，研究原代分离的肝细胞对于确定它们在肝脏生理学和病理生理学中的作用是必要的。特别是，分离细胞的数量和质量对其功能至关重要。我们的目的是从单个肝脏标本中分离大量高质量的人实质和非实质细胞。通过胶原酶灌注结合低速离心、密度梯度离心和磁激活细胞分选从肝组织中分离肝细胞、枯否细胞、肝窦内皮细胞和星状细胞。通过测定培养细胞群的形态、区分性细胞标志物表达和功能活性来控制培养细胞群的纯度和功能。每克肝组织的细胞制备得到以下细胞计数：2.0±0.4×107个肝细胞、1.8±0.5×106个枯否细胞、4.3±1.9×105个肝窦内皮细胞和3.2±0.5×105个星状细胞。通过白蛋白（95.5±1.7%）鉴别肝细胞，并显示细胞色素P450酶的时间依赖性活性。Kupffer细胞表达CD 68（94.5±1.2%），并显示吞噬活性，用1μm乳胶珠测定。内皮细胞为CD 146+（97.8±1.1%），并表现出对乙酰化低密度脂蛋白的有效摄取。α-平滑肌肌动蛋白（α-smooth muscle actin）阳性率为97.1±1.5%。这些细胞进一步表现出视黄醇（维生素A）介导的自发荧光。我们的原代肝实质细胞和非肝实质细胞的分离程序产生了高纯度和高质量的细胞群，并保留了体外生理功能。因此，该系统可以提供用于确定肝功能和疾病的有价值的工具。
Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×107 hepatocytes, 1.8±0.5×106 Kupffer cells, 4.3±1.9×105 liver sinusoidal endothelial cells, and 3.2±0.5×105 stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146+ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.