我们要感谢李嘉刘为他们的创作支持图1的这项研究是由教育与研究部（BMBF）项目虚拟肝德国联邦卫生部的支持:0315741。

注:所有细胞从手术非肿瘤性人肝组织，这仍然偏后肝切除原发或继发肝肿瘤隔离。 Universitätsmedizin柏林 - 根据查理特的道德准则，得到患者的知情同意。 1.材料和解决方案的研制<ol><li>消毒所有仪器和材料事先避免在隔离过程细菌污染。 </li><li>制备用于肝组织样品，肝细胞和非实质肝细胞的分离方法，并根据表1和2中的原代人肝细胞培养的灌注所需的解决方案，这是新鲜制备的消解解的例外在使用之前。所有的解决方案可以被存储在4℃下，建议制备后4周内使用它们。 </li><li>消毒采用0.22微米瓶顶过滤器的所有解决方案。 </li></ol> 2.灌注设备的研制<ol><li>设置的设备的肝组织样品的灌注和消化， 如图1A所示 。 </li><li>调节水浴温度至39℃，以确保灌注和消化过程中最佳胶原酶P活性。 </li></ol>肝组织样品的3灌注和消化（1.5小时） <ol><li>选择与来自切除肝组织完整Glisson囊的组织样品。当切割组织样本，尝试得到具有良好可见血管的小的切割表面。在运输和冰上处理肝脏组织样本，直到血流灌注，避免热缺血时间。 </li><li>取组织重量在无菌条件下，并放置在肝组织样品中的空气层流培养皿。清洁组织样品的表面与来自重无菌压缩剩余的血，并使用1×灌注-溶液I，以确保所有的套管都是可渗透冲洗套管组。 </li><li>使用组织胶固定套管的橄榄在一些较大的血管。取决于肝组织样品的大小和表面上的血管的数量，使用的插管具有3至8的插管设置。测试灌注及检查泄漏。关闭所有的血管，从而泄露明确1X灌注的解决方案我，用纸巾胶水。 </li><li>放置插管肝组织样本进入其穿孔的过滤盘（ 图1A）上的布氏漏斗。 </li><li>置7.5毫升/分钟，这取决于所用的插管的数目和对肝脏组织的电阻14.6毫升/分钟之间的蠕动泵的流速。每次调节流速，以确保有电流通过，但缓慢灌注。灌注所述组织，直到全血冲洗出来，但至少20分钟。观察组织具有良好的perfusi区域变得更亮上。 注意:在某些情况下，可能有必要以夹紧用塑料夹具插管之一或通过与针对肝包膜抹刀轻轻推动以增加面积的内压，以优化灌注。一个完整的颜色更改为浅黄色至浅棕色的颜色表明了良好的灌注。 </li><li>改变灌注流体含有胶原酶P（ 表1）消化-解。 </li><li>重新排列为消化步骤的安装（ 图1A）。因此，根据图1B进行长达15分钟消化溶液的循环流动。 注:关键是要立即停止灌注时肝组织样本被充分消化。一个好的消化可以观察到，当组织没有显示出弹性的迹象由维修胶囊变形的评估中，当用刮刀推动。 </li></ol> 4.隔离肝细胞（1小时） <ol><li> ŧ瓮蠕动泵关闭并将肝组织样品中的玻璃培养皿中。冲洗组织样品的外部用冰冷的停止溶液（ 表1）。取下肝脏组织样本插管。使用解剖刀打开肝组织样品，通过在将插管附着的区域的中间切开。请注意使Glisson&#39;s胶囊保持不变。 </li><li>冲洗组织样品的内部再涂用冰冷的停止 - 解整个组织样品。轻轻摇动组织释放出的细胞组织的。 </li><li>收集细胞悬浮液，并通过一个视线漏斗（塑料漏斗用纱布压缩衬里）到50ml塑料管中过滤。添加更多的停止 - 解肝组织样品，直到500 ml的终体积被消耗。 </li><li>离心细胞悬浮液，在50 XG，5分钟，4℃。收集后非实质细胞分离上清。用PBS（ 图2洗细胞沉淀A）。 </li><li>在50 XG，5分钟，4℃再次离心细胞悬浮液。收集上清液并重新悬浮在肝细胞孵育培养基中的小球（ 表2， 图2B）。 </li><li>确定在使用台盼蓝染色得到的细胞悬浮液中的细胞数量和生存力。算上在Neubauer计数室的生活和死亡的细胞。计算PHH的使用下面的公式细胞数，存活力和产量。 细胞悬液（毫升）的产量（计数细胞）=计数细胞×稀释系数x数量×万 产率（肝/（每克肝组织样品））=（细胞悬浮液（毫升）产率（肝/（毫升培养基））×体积）/（重量肝组织样品（G）） 存活率（％）= 100％×（数活细胞）/（总细胞数） </li></ol> 5.肝细胞的纯化（1小时） 注:建议该纯化步骤中，如果存活力是低于70％。 <ol><li>执行冰的所有步骤。通过混合5毫升密度梯度溶液和15ml PBS中密度梯度离心制备的25％的密度梯度。 </li><li>小心缓慢地把一个最大50宇细胞共出肝细胞丰富细胞悬浮在25％的密度梯度层的顶部，以确保这两个层的一个明确分开实现（ 图2C）。小心翼翼地把管放入离心机离心1250 XG，20分钟，4℃不带刹车（ 图2D）。 </li><li>吸出剩余的细胞悬浮液，并在相间的死细胞。取决于脂肪内容的一个可能也吸出密度梯度的溶液。 注:PHH低脂肪含量形成致密颗粒和密度梯度可以完全被吸出。 PHH具有高脂质含量形成更弥漫粒料和大量活细胞可保留在颗粒上面的密度梯度溶液中。 </li><li>在50 XG，5分钟，4℃再次重新悬浮，用PBS离心肝细胞粒料。池小球，用PBS洗一遍，并在肝细胞培养液重新悬浮纯化PHH。如步骤4.6中所述进行细胞计数。 </li></ol> 6.培养肝细胞<ol><li>通过用细胞外基质涂覆它们，例如鼠尾胶原（I型胶原）准备PHH的播种的细胞培养皿。根据由拉詹等人建立的协议准备的鼠尾胶原。17 </li><li>在PBS 200的稀释:鼠尾胶原蛋白原液1。转移100微升/厘米2鼠尾胶原溶液放入培养皿，照顾整个表面被覆盖。孵育细胞培养物塑料在室温下20分钟。吸剩下的鼠尾胶原溶液。 </li><li>种子15×10 4肝细胞/厘米2的肝细胞培养液对文化dishe包被的有鼠尾胶原蛋白。培养在湿润的培养箱将细胞在37℃，5％CO 2的至少4个小时。 4小时后的肝细胞已经粘附和介质是可以改变的。 </li><li>根据使用的实验装置的调查。建议48小时的培养时间，以使细胞从隔离过程中恢复。 </li></ol> 7.隔离非实质肝细胞（1.5-2小时） <ol><li>离心机72 XG，5分钟，4℃收集的上清液（步骤4.5和4.6），以消除剩余的红细胞和肝细胞。池上清液并离心他们两次以获得两个细胞丸粒:300 XG，5分钟，4℃的HSC，LEC和部分的KC和650 XG，7分钟，4℃对剩余的KC沉降的沉淀。 </li><li>在HBSS池既颗粒和重新挂起他们。制备25％，通过将密度梯度溶液和PBS对密度梯度centrif 50％的密度梯度ugation（25％的密度梯度溶液分配:5ml密度梯度溶液和15ml的PBS，50％的密度梯度溶液:10毫升密度梯度溶液和10ml的PBS，参见图2）。小心地将25％的密度梯度溶液的50％的密度梯度溶液层的顶部。 </li><li>小心缓慢地把全国人民代表大会停牌的25％的密度梯度溶液层的顶部，这两个层的明确分离实现的一种方式。 </li><li>在离心机在1800 XG，20分钟，4℃不带刹车（ 图2.2）密度梯度的细胞悬液。 </li><li>吸死细胞和从最上层的细胞碎片。人大位于25％和50％的密度梯度层（ 图2）之间的间期。收集鼻咽癌，用HBSS和离心机洗应用上述双离心步骤的细胞悬浮液（步骤7.2）。 </li></ol> 8.分离枯否细胞（粘附分离工序）（1小时） <ol><li>如步骤4.6中所述进行该KC在NPC馏分细胞计数。 （对于悬浮KC的外观见图3B）。离心机人大馏分用上述双离心步骤（步骤7.2）和重新悬浮人大在枯否细胞接种培养基（ 表2）。 </li><li>种子以5×10 5的KC / cm 2的密度在塑料细胞培养容器对KC含有级分。孵育KC培养物在加湿培养箱中20分钟，在37℃，5％ 的 CO 2。主KC附着在细胞培养物塑料的短时间（ 图2.3）之内。 </li><li>收集含有不遵守NPC上清为主的LEC和HSC。池LEC以后分离上清液（见第9）和HSC（见第10）。用HBSS洗粘附KC，并在37℃，5％CO培养他们在枯否细胞培养基（ 表2）2在加湿培养箱。 </li></ol> 9.分离内皮细胞（1.5小时） <ol><li>离心机在300 xg离心，5分钟，4℃收集的上清液（步骤8.5）。用PBS洗沉淀。在300 XG，5分钟，4℃再悬浮在星状细胞/内皮细胞分离培养基中的细胞，并如在步骤4.6中描述的所有剩余细胞进行细胞计数离心后。 </li><li>重悬在1毫升星状细胞/内皮细胞分离液1×10 7神达细胞，加入20微升来自MACS-KIT封闭液和20微升的CD31微珠为免疫标记，并在4孵育所得的悬浮液15分钟°C的温度（ 图2.4）。 </li><li>作为磁激活细胞分选系统MACS（ 图2.5）在manufacturer&#39;s议定书中所描述的HSC单独LEC。磁洗脱保留CD31阳性LEC和星状Ç他们暂停ELL /内皮细胞培养基（ 表2）。 </li><li>如步骤4.6中所述进行细胞的计数LEC。在1.25×10 5细胞/ cm 2的涂有鼠尾胶原细胞培养容器的密度种子LEC（参见步骤6.1）。培养细胞在37℃，5％CO 2在加湿培养箱。 </li></ol> 10.分离星状细胞（0.5小时） <ol><li>未标记的HSC的MACS过程中通过分离柱。收集HSC分数（见步骤9.5， 图2.5）。如步骤4.6中所述进行细胞计数。 </li><li>用5×10 4个细胞/ cm 2的细胞培养容器涂有鼠尾胶原的密度种子HSC（参见步骤6.1）在星状细胞/内皮细胞培养基（ 表2），并在37℃，5％加以培养的 CO 2在加湿培养箱。 </li></ol><img alt="表1"SRC ="/文件/ ftp_upload / 53069 / 53069table1.jpg"/&gt; 表1: 灌注和隔离解决方案。 <img alt="表2" src="/files/ftp_upload/53069/53069table2.jpg" /> 表2: 培养和分离介质。

<ol>
	<li>Pfeiffer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+cultivation+of+human+hepatocytes+and+non-parenchymal+liver+cells.">Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells.</a> Exp Biol Med. , (2014).</li><li>Si-Tayeb, K., Lemaigre, F. P., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+and+development+of+the+liver.">Organogenesis and development of the liver.</a> Dev Cell. 18 (2), 175-189 (2010).</li><li>Alpini, G., Phillips, J. O., Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+isolation+of+liver+cells.">Recent advances in the isolation of liver cells.</a> Hepatology. 20 (2), 494-514 (1994).</li><li>Godoy, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+2D+and+3D+in+vitro+systems+using+primary+hepatocytes,+alternative+hepatocyte+sources+and+non-parenchymal+liver+cells+and+their+use+in+investigating+mechanisms+of+hepatotoxicity,+cell+signaling+and+ADME.">Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.</a> Arch Toxicol. 87 (8), 1315-1530 (2013).</li><li>G&#243;mez-Lech&#243;n, M. J., Castell, J. V., Donato, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocytes--the+choice+to+investigate+drug+metabolism+and+toxicity+in+man:+in+vitro+variability+as+a+reflection+of+in+vivo.">Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo.</a> Chem Biol Interact. 168 (1), 30-50 (2007).</li><li>Ginai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+bioreactors+as+in+vitro+models+in+pharmaceutical+research.">The use of bioreactors as in vitro models in pharmaceutical research.</a> Drug Discov Today. 18 (19-20), 922-935 (2013).</li><li>Schyschka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+3D+cultures+but+not+2D+cultures+preserve+specific+transporter+activity+for+acetaminophen-induced+hepatotoxicity.">Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity.</a> Arch Toxicol. 87 (8), 1581-1593 (2013).</li><li>Kostadinova, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long-term+three+dimensional+liver+co-culture+system+for+improved+prediction+of+clinically+relevant+drug-induced+hepatotoxicity.">A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity.</a> Toxicol Appl Pharmacol. 268 (1), 1-16 (2013).</li><li>Messner, S., Agarkova, I., Moritz, W., Kelm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-cell+type+human+liver+microtissues+for+hepatotoxicity+testing.">Multi-cell type human liver microtissues for hepatotoxicity testing.</a> Arch Toxicol. 87 (1), 209-213 (2013).</li><li>Nussler, A. K., Nussler, N. C., Merk, V., Brulport, M., Schormann, W., Yao, P., Hengstler, J. G., Santin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Holy+Grail+of+Hepatocyte+Culturing+and+Therapeutic+Use.">The Holy Grail of Hepatocyte Culturing and Therapeutic Use.</a> Strategies in Regenerative Medicine. , 1-38 (2009).</li><li>Friedman, S. L., Roll, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hepatic+lipocytes,+Kupffer+cells,+and+sinusoidal+endothelial+cells+by+density+gradient+centrifugation+with+Stractan.">Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan.</a> Anal Biochem. 161 (1), 207-218 (1987).</li><li>Knook, D. L., Blansjaar, N., Sleyster, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Kupffer+and+endothelial+cells+from+the+rat+liver.">Isolation and characterization of Kupffer and endothelial cells from the rat liver.</a> Exp Cell Res. 109 (2), 317-329 (1977).</li><li>Alabraba, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+isolation+and+culture+of+human+Kupffer+cells.">A new approach to isolation and culture of human Kupffer cells.</a> J Immunol Methods. 326 (1-2), 139-144 (2007).</li><li>Friedman, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+hepatic+lipocytes+and+Kupffer+cells+from+normal+human+liver:+morphological+and+functional+characteristics+in+primary+culture.">Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.</a> Hepatology. 15 (2), 234-243 (1992).</li><li>Lalor, P. F., Lai, W. K., Curbishley, S. M., Shetty, S., Adams, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+sinusoidal+endothelial+cells+can+be+distinguished+by+expression+of+phenotypic+markers+related+to+their+specialised+functions+in+vivo.">Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo.</a> World J Gastroenterol. 12 (34), 5429-5439 (2006).</li><li>Lee, S. M., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+hepatocytes+by+a+two-step+collagenase+perfusion+procedure.">Isolation of human hepatocytes by a two-step collagenase perfusion procedure.</a> J Vis Exp. (79), (2013).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat Protoc. 1 (6), 2753-2758 (2006).</li><li>Chang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hepatic+stellate+cells+from+mouse+liver.">Isolation and culture of hepatic stellate cells from mouse liver.</a> Acta Biochim Biophys Sin (Shanghai). 46 (4), 291-298 (2014).</li><li>Zeng, W. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+to+isolate+and+culture+rat+kupffer+cells.">A new method to isolate and culture rat kupffer cells.</a> PLoS One. 8 (8), e70832 (2013).</li><li>Tokairin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+specific+isolation+of+rat+sinusoidal+endothelial+cells+by+the+immunomagnetic+bead+method+using+SE-1+monoclonal+antibody.">A highly specific isolation of rat sinusoidal endothelial cells by the immunomagnetic bead method using SE-1 monoclonal antibody.</a> J Hepatol. 36 (6), 725-733 (2002).</li><li>Damm, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+parenchymal+and+non-parenchymal+liver+cell+isolation,+culture+and+characterization.">Human parenchymal and non-parenchymal liver cell isolation, culture and characterization.</a> Hepatology International. 7, 915-958 (2013).</li><li>Baccarani, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+hepatocytes+from+livers+rejected+for+liver+transplantation+on+a+national+basis:+results+of+a+2-year+experience.">Isolation of human hepatocytes from livers rejected for liver transplantation on a national basis: results of a 2-year experience.</a> Liver Transpl. 9 (5), 506-512 (2003).</li><li>Shen, L., Hillebrand, A., Wang, D. Q., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> J Vis Exp. (64), (2012).</li><li>Gerlach, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+isolation+of+sinusoidal+endothelial+cells+from+pig+and+human+liver.">Large-scale isolation of sinusoidal endothelial cells from pig and human liver.</a> J Surg Res. 100 (1), 39-45 (2001).</li></ol>

The authors declare that they have no competing interests.

已发布的协议描述的技术来分离纯PHH和NPC，即KC，HSC和LEC，在从人肝脏组织的相同样品的高质量和纯度同时。大多数处理肝细胞隔离出版物只包括与人体组织进行的细胞群18-20和分离方法之一是罕见的21条 （达姆等回顾）。与动物组织（如大鼠肝脏），以人类肝脏建立的方法适应揭示了动物和人类细胞群之间的单元格属性一些差异。覆盖不同的肝细胞群体肝细胞的分离方法的建立显示，组合实质和非实质细胞分离是关键的一步，由于在用于优化的结果所需的消化时间差。面对这一挑战，我们开发了一种肝细胞分离协议结合不同的技术和适应了本身我们PHH隔离协议10。 通过加入10％FCS的含有溶液II胶原酶的P，我们能够降低蛋白酶的蛋白水解活性。此修改允许更长的消化时间，必需取得高数量的NPC。其结果是，我们能够隔离PHH以及质量好全国人大和高量。成功的肝细胞分离初始质量组织的强烈依赖。供体的数据和病历可以影响细胞的质量和数量。根据我们的经验有一个与捐赠者的具体因素无相关性，也非常有经验的工作人员可以面对失败的细胞分离。因为供体组织的质量是一个临界点，所有的外部源，这可能损害组织的质量，必须被最小化。 最关键的因素是手术干预和冷缺血时间后热缺血时间的组织T运输过程中澳实验室。此外，应避免对细菌感染的任何来源。它必须注意的是，有时该器官本身可以包含细菌污染， 例如，在胆汁道疾病的情况下。分离过程中的关键步骤盖住灌注时间和密度梯度离心的步骤。第一灌注步骤应持续20-30分钟。缩短灌注时间可能导致的从而在获得细胞悬浮液的细胞簇的发生细胞 - 细胞接触不完全脱离。长时间的第一灌注降低细胞活力和诱导细胞应激由于钙离子耗竭。 用于酶组织消化进行第二灌注步骤需要一些经验来确定采取有关停止在合适的时间点上的蛋白水解反应决定最佳的消化程度。短的消化时间导致低收益率和延长消化时间为细胞应激和细胞损伤。从我们的经验，在消化过程中未消化的组织和受损组织之间的时限往往在于1-3分钟的窗口内。一个不完整的灌注可以通过改变组织内的压力成使用夹具为夹断的插管另一区域被抵消。朝向组织样品抹刀此外软压力导致在组织灌注的变化。所述组织是压缩和内部压力增加。随后，血管也被压缩和其半径减小。在半径减小，阻力增大（哈根 - 泊肃叶定律）和灌注溶液倾向于阻力最小和perfuses其他区域的方式。按照Baccarani和同事我们观察到纤维化或肝硬化的组织需要一个较长的消化期间，导致降低细胞存活率22。为此，我们建议，以避免患者肝纤维化或肝硬化的组织。 制备和密度梯度处理，以及收获细胞出梯度（见步骤5和7）也包括关键步骤。不同的密度梯度层和细胞悬浮液在一个缓慢而小心的方式被传输创造尖锐间期。此外，还有总的处理过程中损坏梯度，尤其是在收获细胞的风险。在NPC分离的贴壁分离步骤是以后的产量和纯度的所有NPC分数的关键。为了提高不遵守NPC的细胞数量和KC的纯度额外的洗涤步骤可以是有益的。该步骤的洗涤液被合并与上清液用于进一步的NPC分离。以避免由细菌污染，真菌或病毒严格的无菌条件下，必须确保23。 在所需的细胞群的依赖性的协议可以通过跳过特定的步骤来改变和调整。例如，如果仅KC是需要鼻咽癌沉淀的双重离心步骤可以降低到第二离心分离步骤和用于HSC和LEC分离步骤可以被丢弃。灌注时间和G力的离心分离，也可以在组织和细胞质量的依赖性变化。纤维化组织需要延长的消化时间，因此，有必要仔细地控制组织弹性。脂肪肝脂滴的累积降低了细胞密度和因此改变沉降特性。根据我们的观察它可以是有用的视PHH，需要高PHH量时的脂质含量中PHH隔离调整的g力。它必须指出的是，初始离心步骤的任何更改将负在质量和数量方面的人大隔离影响。到目前为止，我们建议50 XG（脂肪含量低的肝细胞）和150 XG（脂肪含量高的肝细胞）之间的G力。此外脂肪HEPAtocytes倾向于形成密度梯度离心后较少紧凑细胞沉淀和进一步细胞收集具有如步骤5.5中所述进行改变。 加快分离过程的一些步骤可以同时进行。例如与分离的肝细胞的纯化平行的第二人可以与NPC隔离开始。另外，该密度梯度溶液可提前制备。如果有两个以上的人甚至更多个步骤可以同时进行。 相较于其他人肝细胞的分离协议，我们的研究结果显示出类似的或更高的细胞产率和存活率如前文实验生物及医学出版1。为KC隔离Alabraba和同事证明了隔离的结果为2.3×10 6个活KC / g的肝组织的约98％13生存力，这是相当于组合的产量我们的KC的结果（细胞数:1.9×10 6个活KC / g的肝组织，存活率约为93％）。多数公布的LEC隔离的数据描述了从整个器官15,24隔离。格拉赫和同事以及拉洛尔和同事分离10 3和10 6个细胞/器官15,24之间的细胞数。这些数据不能直接比较，细胞从组织样品隔离。然而，用我们的协议中，我们表现出产量为2.7×10 5可行的LEC / g的肝组织，在全器官推断当这是目前较大的LEC。 HSC与约4.7×10 5个存活的HSC / g的肝组织的产率和生存能力约90％分离。由Friedman和同事发表现有结果表明半低级细胞产量（2.3×10 5的HSC / g的肝脏），但类似的纯度（91％）14。关于我们的协议中，低产量的细胞可以通过灌注不良和消化造成的，由于1X灌注搜索解决方案的低或无流通n个I和组织内消化解决方案。此外，在该组织中的气泡可以扰乱1X灌注的解决方案我和消化的解决方案的组织内循环。在这些情况下，灌注可以通过夹紧单一插管和/或使用一个抹刀推靠在组织样本增加气泡的灌注压和消除得到改善。一个坏的生存能力是在大多数情况下，细胞应激的结果。长时间缺血时间，由钙离子耗竭和膜蛋白的水解损坏都与细胞损伤，由细胞膜泡可见。从我们的观察这些细胞一个非常敏感的剪切应力，并在大多数情况下，分离过程中死亡。总之，成功地分离和实质和非实质肝细胞的分离需要关键步骤是在正确的时间帧进行的，被用于细胞的分离和分离小号仔细和一般执行移液步骤的时间HOULD保持尽可能短21。所描述的协议的一个缺点是，在隔离条件（ 例如 ，灌注次）不能完全标准化，但也可以个别地适应于组织质量。此外，获得的细胞群的产率和纯度可在组织的质量和消化结果而变化。 我们最近发表的一项研究表明栽培条件，用这种方法1分离的每个NPC细胞类型的功能特性相结合的影响。的可能性来分离和肝脏的分离的不同细胞群体可具有革新性人肝细胞的共培养和组织工程的体外肝模型创建。这是众所周知的，在2D单培养PHH栽培导致去分化和典型的细胞功能7的损失。出于这个原因，有必要模仿体内组织ARCHI体外肝脏模型中tecture。科斯塔迪诺娃和同事（2013 8,9）以及梅斯纳尔和同事（2013 8,9）成功建立官能共培养肝模型用于检测肝毒性作用。然而，鼻咽癌均未特征和具体功能并没有在这些系统的影响。 因此，进一步的研究应着眼于共同文化中对鼻咽癌的长期生存调查，其具体特点和相互作用。对于这样的研究，也有可能是感兴趣的建立一个协议，用于胆管的隔离。官能体外共培养物，包括载于天然肝所有细胞类型的实现可能是一个进一步的步骤到在体内像人类肝脏模型的方向。

人肝组织是高度复杂的，由两个不同的细胞的实体，实质细胞和非实质细胞（NPC）的。实质肝细胞包括肝细胞和胆管。肝细胞代表总肝细胞的60至70％，占大部分的代谢肝功能， 例如，胆汁酸和补体因子的合成，生物转化和能量代谢2,3。 较小的人大馏分构成总肝细胞的30​​-40％。鼻咽癌包括不同的细胞群，即Kupffer细胞（KC），肝内皮细胞（LEC）和肝星状细胞（HSC）。此异种细胞部分起着在肝脏的生理过程中心作用。此外，NPC参与调解急性肝损伤， 如药物性肝损伤（DILI）以及慢性肝损伤，如肝硬化4。 近年来，高度乌曼肝细胞在肝脏疾病已成为研究和药物测试，药物开发和新的生化途径鉴定发展，越来越多的关键。对于体外试验PHH单一种植仍被认为是"黄金标准"5。电流同型肝模型的主要限制是在几天内4去分化和肝细胞功能的丧失。的3维（3D）培养技术建立已经表明这些限制可以补偿4,6。然而，即使现代3D培养技术是无法显示的操作7所有肝毒性模式。缺少鼻咽癌种群在现有的体外模型作为一个可能的原因这种差异的体内情况进行讨论。已经表明，不同的肝细胞群体之间的细胞 - 细胞通讯起着生理动态平衡中发挥中心作用，而且在pathophysiologic处理8。因此，科学的关注重点越来越多的NPC和细胞间的相互作用。它们在共培养和组织工程化的系统目的的用途可能是用于体外肝模型8,9-这是因为接近尽可能体内情况的高需求的解决方案。 目前的主要挑战是标准化的人类肝脏共培养模型，其中包含PHH和NPC的明确规定部分的发展。结果，需要为非常异种肝细胞的分离技术和那些必须被优化以获得纯的细胞群。虽然有10 PHH隔离标准化的协议，人类NPC的标准化隔离仍在发展。大多数公布鼻咽癌隔离协议是基于与非人类细胞11,12实验。只有少数出版物描述人类鼻咽癌的隔离工艺和最多只是覆盖键入11-16用于单个细胞的分离方法。已被利用用于细胞分离的最重要的细胞特征是尺寸，密度，附着行为，和表面蛋白的表达。在这些特征的基础上，我们开发了一个简单的协议来隔离PHH，KC，LEC和HSC，这是在实验生物及医学 1先前公布。因为在这种技术的广泛兴趣的，本文的目的是提供用于肝细胞分离过程包括视频，这将允许更容易地再现的技术中的更​​详细的协议。该协议还包括用于产量和活力的评价以及利用特定immunostainings鉴定和纯度评估质量控制方法。

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">General Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:293px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PIPETBOY</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2-incubator</td>
			<td>Binder</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lamin Air</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge Varifuge 3.0R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urine Beaker</td>
			<td>Sarstedt</td>
			<td>2041101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">perfusor syringe 50 ml</td>
			<td>B.Braun</td>
			<td>12F0482022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bottle Top Filter</td>
			<td>Nalgene</td>
			<td>1058787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 50 ml Polypropylene Conical Tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 15 ml Polypropylene Conical Tube</td>
			<td>BD Biosciences</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Culture plate</td>
			<td>BD Biosciences</td>
			<td>533047</td>
			<td>24 well</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">serological pipettes</td>
			<td>BD Biosciences</td>
			<td>357525, 357551, 357543</td>
			<td>25 ml, 10 ml, 5 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipette tips</td>
			<td>SARSTEDT</td>
			<td>0220/2278014, 0005/2242011, 0817/2222011</td>
			<td>100 &#181;l, 200 &#181;l, 1000 &#181;l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isolation Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">water bath</td>
			<td>Lauda</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">peristaltic pump</td>
			<td>Carl Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">circulation thermostat</td>
			<td>Lauda</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH meter</td>
			<td>Schott</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fine scales</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B&#252;chner funnel</td>
			<td>Haldenwanger</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">plastic funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silicone tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cannulae with olive tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scalpel</td>
			<td>Feather</td>
			<td>12068760</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neubauer counting chamber</td>
			<td>Optic Labor</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cell lifter</td>
			<td>Costar</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Drape</td>
			<td>Charit&#233; Universit&#228;tsmedizin Berlin</td>
			<td>A2013027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">compress</td>
			<td>Fuhrmann</td>
			<td>40013331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sterile surgical gloves</td>
			<td>Gammex PF</td>
			<td>1203441104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue glue</td>
			<td>B. Braun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass bottle</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche</td>
			<td>13349524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Percoll Separating Solution</td>
			<td>Biochrom</td>
			<td>L6145</td>
			<td>Density 1.124 g/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s BSS</td>
			<td>PAA</td>
			<td>H00911-3938</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#8217;s PBS</td>
			<td>PAA</td>
			<td>H15 - 002</td>
			<td>without Mg/Ca</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampuwa</td>
			<td>Plastipur&#160;</td>
			<td>13CKP151&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Merck</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Merck</td>
			<td>49,361,000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Pufferan&#160;</td>
			<td>Roth</td>
			<td>133196836</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E-5134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Media Equipment&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>PAA</td>
			<td>E15-005</td>
			<td>Low Glucose (1 g/l) (without L-Glutamine)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer Solution 1M</td>
			<td>GIBCO</td>
			<td>1135546</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>GIBCO</td>
			<td>25030-024</td>
			<td>200 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM NEAA</td>
			<td>GIBCO</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>PAA</td>
			<td>E15 - 039</td>
			<td>without L-Glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>GIBCO</td>
			<td>1137663</td>
			<td>100 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue Solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>0.4%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">William&#8217;s E&#160;&#160;</td>
			<td>GIBCO</td>
			<td>32551-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">with GlutaMAX&#8482;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>03780-50G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fortecortin</td>
			<td>Merck</td>
			<td>49367</td>
			<td>8 mg/2 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human-Insulin</td>
			<td>Lilly</td>
			<td>HI0210</td>
			<td>100 I.E./ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal calf serum (FCS)</td>
			<td>PAA</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment for Immunostainings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD 68</td>
			<td>R&#38;D Systems, USA</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK 19</td>
			<td>Santa Cruz</td>
			<td>D2309</td>
			<td>polyclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK18</td>
			<td>Santa Cruz</td>
			<td>K2105</td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vimentin</td>
			<td>Santa Cruz</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GFAP</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>monoclonal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>23.472-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse IgG1-PE</td>
			<td>Santa Cruz</td>
			<td>C0712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit IgG-FITC</td>
			<td>Santa Cruz</td>
			<td>L0412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>J.T.Baker</td>
			<td>1104509006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde 4%</td>
			<td>Herbeta Arzneimittel</td>
			<td>200-001-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for isolation of primary human hepatocytes and corresponding major populations of non-parenchymal liver cells

旁肝实质细胞，肝脏由非实质细胞（NPC）即Kupffer细胞（KC），肝内皮细胞（LEC）和肝星状细胞（HSC）的。原代人肝细胞（PHH）的二维（2D）文化仍然被认为是"黄金标准"的体外药物代谢和肝毒性的测试。它是众所周知的，PHH的2D单作从去分化和功能丧失缺点。最近已表明肝鼻咽癌在肝（病理）生理学中心作用和PHH功能的维持。目前研究的重点体内组织架构的重建由3D-共培养模式，以克服单一种植2D的局限性。先前我们发表以分离的人肝细胞的方法，并研究这些细胞的适合其在细胞培养物中的实验生物学和医学1使用。基于此TE的广泛兴趣chnique本文的目的是提供用于肝细胞分离过程包括视频，这将允许该技术的一个简单的再现的更详细的协议。 人肝细胞从外科手术的人肝组织样品通过一个两步EGTA /胶原酶P灌注技术分离。 PHH从人大通过的初始离心50×g的分离。被用于去除死细胞的密度梯度离心的步骤。个人肝细胞群体从富集的人大分数使用特定的电池特性和细胞分选程序中分离。旁边的PHH隔离，我们能够分离KC，LEC和HSC进一步培育。 总之，所提出的协议允许PHH和NPC的从一个供体组织样本的高品质和数量的隔离。纯化的肝细胞群的访问可以让人类像李创造体内版本车型。

分离成一个实质和非实质级分，使用密度梯度离心作为清理过程与使用的附着性和MACS的结合导致成功PHH和NPC隔离。 PHH和NPC可以在高的质量和数量进行分离。 图1示出了设备的肝灌注和消化的代表性设置。 10％FCS中加入含灌注胶原酶对 - 溶液II降低蛋白酶的蛋白水解活性，并在返回到稳定胶原酶P活性。在鼻咽癌隔离所需结果更长的消化时间可以在不PHH的可行性产生负面影响被应用。 <img alt="图1" src="/files/ftp_upload/53069/53069fig1.jpg" /> 图 1: 灌注和消化设置的第一灌注步骤是在奥德进行R以除去残留的血液，热身组织并除去的 Ca 2+通过使用1倍灌注溶液I（PI）（A）中，以溶解细胞-细胞连接。期间灌注步骤II（B）为肝组织进行消化消解-解决方案（DG）的再循环。 <img alt="图2" src="/files/ftp_upload/53069/53069fig2.jpg" /> 图2:。 完整的PHH和NPC隔离工艺简图表示从Pfeiffer 等 1 2014改性实验生物及医学的许可。首先，将肝组织样品灌注和通过一个两步EGTA /胶原酶P灌注技术（A）的消化。收获的细胞悬浮液在50 XG，5分钟，4℃（B）中，以从更小的人大馏分较大PHH-级分（沉淀）分离（supern最初离心atant）。在70％以下一个PHH生存能力的情况下，可行的PHH分数可以通过密度梯度离心在1250 xg离心，20分钟，4℃（℃）导致在管的底部的PHH沉降富集，而死细胞/细胞碎片位于密度梯度层（D）的顶部。 1）300 XG，5分钟，4℃，2）650 XG，7分钟，4℃:初始离心（1）的收集的上清液用两步离心。第一次离心后的KC部分地位于在上清液中。在这种情况下，第二分离步骤是必要的。收获的细胞沉淀物合并并重新悬浮于HBSS中。随后，将细胞悬浮液仔细地层叠在两层（25％/ 50％）的密度梯度的顶端。层状密度梯度管在1800 xg离心，20分钟，4℃离心分离（2）。在25％的密度梯度层的顶部死细胞被丢弃。鼻咽癌位于的25％的间期和50％d个之间密度梯度层被收集并汇集。全国人大分数接种在无涂层的细胞培养塑料。使用20分钟的温育时间（附着分离工序）KC从其它肝细胞群体分离（3）。 LEC和HSC通过使用MACS试剂盒分离。因此在上清液收集的剩余肝细胞以300 xg离心，5分钟，4℃离心分离，并贴有CD31缀合的微珠（4）。只有CD31阴性HSC通过MACS分离柱（5）。 CD31阳性LEC粘到柱上。最后，列从磁器件和CD31阳性LEC删除洗脱出列（5）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53069/53069fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 孤立PHH显示，14.2×10 6±6.6×10 6可行PHH / g的肝组织的收率和生存能力约760.6±4.2％（ 表3 1）。镜下可见特征是在组合的典型的大型细胞质体积与脂滴和核1和4之间，（ 图3A）。该单元尺寸悬浮20 30之间变化微米。 KC是在NPC馏分中最常见的细胞类型。我们分离大约1.9×10 6±0.2×10 6个活KC / g的肝组织的92.8±3.5％（ 表3 1）生存力。 KC是非常小的细胞（约5μm）用低细胞质/细胞核比例和典型微绒毛表面（ 图3B）上。 最后，我们使用的MACS分离技术的CD31阳性LEC从剩余的CD31阴性HSC分离。 LEC的产率是约2.7×10 5±0.1×10 5个存活的LEC / g的LIV器组织和实现存活率为95.6±2.8％（ 表3 1）。鉴定标准是多个granulae和短培养时间（ 图3G）后的大小为在细胞悬浮液（ 图3C），以及特性纺锤形约10微米。 隔离过程产生具有89.6±3.8％（ 表3 1）一个生存能力的HSC收率约4.7×10 5±0.2×10 5个存活的HSC / g的肝脏（N = 8）。微观识别的特性分别为约20微米的尺寸和典型的造粒外观脂滴（ 图3D）的变化的量。 <img alt="表格1" src="/files/ftp_upload/53069/53069table3.jpg" /> 表3: 产量，可行性和孤立PHH和N的纯度个人电脑，三个不同供体进行了评价。数据被表示为平均值±标准差。此表在Pfeiffer 等。1，在2014年之前发布，转载来自实验生物及医学许可。 用于鉴定和细胞培养纯度的测定，每分离的细胞级分与抗细胞类型特异性抗原的抗体治疗。将细胞用荧光二级抗体处理并通过免疫荧光显微镜分析。的纯度通过相对于由Hoechst染色显现细胞总数计数阳性荧光染色的细胞来确定。 种植后的PHH 24小时表现出的特征多边形，经常多倍（ 图3E）。 PHH为阳性对照18（ 图3I），并显示出92.3±3.2％的纯度（ 表3 1）。 KC上的细胞培养塑料表面在20分钟内附着。后观察24小时小圆细胞具有突出的圆形细胞核的潜伏期时间（ 图3F）。表面蛋白CD68被用来确定KC（ 图3J）。 CD68阳性细胞的纯度达81.0±5.4％（ 表3 1）。 尽管全国人大分离过程中使用CD31标记的MACS分离它仍然有可能弄脏隔离带LEC CD31。因此，分离培养LEC用CD31染色鉴定和纯度检测。此外LEC具有免疫活性的间充质细胞标志物波形蛋白（ 图3K）。我们观察到大约阳性染色细胞（ 表3 1）的81.0±1.7％。 </p&gt; HSC与他们的典型突出的脂滴（ 图3H），免疫荧光染色GFAP（ 图3L）标记。 HSC纯度为93.0±1.7％（ 表3 1）。 每个细胞部分与其他NPC标记染。所有的细胞级分包含少量其他肝特定细胞类型的，但均为阴性肝细胞标记物CK18和胆管标记CK19。 <img alt="图3" src="/files/ftp_upload/53069/53069fig3.jpg" /> 图 3: 人类实质和非实质肝细胞悬浮和贴壁后形态左栏（A - D）直接隔离处理后显示了不同的分离肝细胞群在相差显微镜观点:PHH（A），KC（B），LEC（C）和HSC（D）。中间一列（E - 高 ）后种植（相差显微镜）的24小时呈现分离培养PHH（E），KC（F），LEC（G）和HSC（H）的图像。不同细胞级分的基于免疫荧光特性示于最后一栏:PHH显示出对肝细胞标记物CK18阳性信号（I，分离后24小时），KC阳性标记CD68（J，分离后24小时）， LEC呈阳性信号波形（K，隔离后72小时）和HSC呈阳性GFAP（L，分离后72小时）。细胞核被用Hoechst染色;放大倍率:400X。从Pfeiffer 等1 2014改性EXPER许可imental生物学和医学。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53069/53069fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells at JoVE.com

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

protocol-for-isolation-primary-human-hepatocytes-corresponding-major

Nanyang Technological University

Research

JoVE Journal

Biology

36.8K 次观看.  Charité University Medicine Berlin. A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

视频: Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

科研

生物学

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

分离人原代瓣膜细胞进行体外疾病建模

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...