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?...

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

36.9K 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

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

科研

发育生物学

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

免疫与感染

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

乙型肝炎病毒感染原代肝细胞和库珀细胞的 "肝片上" 培养

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

生物学

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

用于肝病研究的外周血人肝球

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

生物工程

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

医学

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. ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

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 ...