Name: in vivo liver endocytosis followed by purification of liver cells by liver perfusion
Uploaded: 2011-11-10T12:00:00.000+00:00
Duration: 12 min 35 s
Description: Watch this Scientific Journal Video about In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion at JoVE.com

我们要感谢保罗威格尔在大学的博士。俄克拉何马州30单克隆抗体检测HARE/Stabilin-2受体和她的技术援助，珍妮特威格尔。这项研究是由内布拉斯加大学研究基金资助。

1。肝切除术（PO Seglen 12和R Blomhoff 13 14,15与修改通过） <ol><li>添加10毫升30％的异氟醚聚乙二醇在一个封闭的5.5大号室培养皿中。等待10-15分钟后，异氟醚除了内室创造一个稳定的气氛，这是最佳。 </li><li>老鼠放置在密封腔，并使其成为麻醉。麻醉的全面影响是显而易见的，当动物成为跛行，反应迟钝，和展品深呼吸。 </li><li>放置2毫升异氟醚聚乙二醇在30 mL注射器管底部的一些棉球。 </li><li>放在尿布覆盖的托盘，并将其放置在口鼻注射器管的大鼠在它的后面。 </li><li>立即证实，用70％乙醇润湿腹部深麻醉。不要让动物死亡异氟醚过量。 </li><li>使用绷带剪刀和镊子，揭露整个ABDominal腔。 </li><li>找到腔PORTA，使用产钳，吸引两股的手术丝线或涤纶线（结扎）下腔以上肠系膜分支立即PORTA。 </li><li>撤回镊子和领带一松上手结。 </li><li>使用下面的腔PORTA钳轻轻拉了回来，以理顺静脉，cannulate Insyte Autoguard导管（1.3 GA，18 × 300毫米，BD​​公司）或类似的导管静脉。导管应超越由线程组成的环几个毫米。 </li><li>缩回，删除通过导管释放弹簧加载触发针。血液应该开始渗入指示正确的结扎安置导管。 </li><li>拧紧上手结和安全与另一上手结。 </li><li>要么切断下腔静脉或降主动脉（主动脉abdominalis），让血液循环系统排出。 </li><li>开始冲洗肝脏含氧TBS电视台在37 ° C，移除在20毫升/分钟的流速的血液（漂白）。肝脏变成一个壤土颜色从紫红色。 </li><li>而肝脏是潮红，切除肝脏切掉消化道和结缔组织。重要的是不要划破Glisson囊肝或穿刺。 </li><li>放置在超过一个漏斗，允许缓冲区来收集和再循环的塑料网肝脏。在这一点上的缓冲区不应再循环，但流入废物烧杯。法拉盛不应持续超过10分钟。 （ 图2A）。准备步骤2.1所需的内吞作用的解决方案。 </li></ol> 2。国际化125 - SA - B - HEP（或其他适当的标记配体） <ol><li>至50 ml的RPMI媒体在小烧杯中，加入终浓度为0.05％BSA和0.01 mCi的125 I - SA - B - HEP或其他适当标记的配体的内吞作用。 </li><li>将烧杯的apparatus到允许充氧。 </li><li>关闭泵，开关入口管，然后打开泵在20毫升/分钟。 </li><li>作为标记的RPMI方法肝脏，关闭泵，并让多余的液体在漏斗和油管漏。 </li><li>媒体烧杯放入漏管允许再循环标记的媒体，然后重新启动泵。 </li><li>让液体通过肝脏不超过1小时循环。 </li><li>在此期间内化步骤，确保在肝脏的温度是通过覆盖它，并准备胶原酶消化稳定。 </li></ol> 3。胶原酶消化肝脏消化<ol><li>新鲜溶解并过滤（0.45微米）胶原酶（100毫克/公斤体重大鼠）应添加到缓冲区的总体积不超过60毫升，在37 ° C。量是依赖于管的长度/内部容积，应作相应调整。 </li><li>停泵和交流从媒体烧杯的TBS入口管。 </li><li>冲洗2分钟，在50 mL / min的钙依赖的桥粒细胞路口松动允许肝脏。 </li><li>而肝脏是潮红，交流媒体烧杯中含有胶原酶烧杯仪器。 </li><li>冲洗后，立即开始在流速为20 mL / min的长达15分钟的消化胶原酶在循环回路。由于胶原酶批次不同批号，在消化的数量和时间的变化，需要为每一个新的胶原酶很多优化。胶原酶也是钙依赖。关闭泵和切换缓冲区和天然气线从保温瓶一个合剂B.转移废物容器的污水合剂乙线（ 图2B）。 </li><li>停泵，取下导管和肝脏转移到一盘菜含有20-30毫升缓冲1。 </li><li>剥开Glisson囊肝和动摇的细胞出去了在液体中。 </li><li>由于液体变为不透明的细胞物质，通过100微米过滤通过一个30微米网格，然后到50 mL锥形冰网格传输的细胞。 </li><li>添加更多的缓冲区1到肝脏，并继续摇晃肝基质细胞，网状过滤器和锥形转移到液体。 </li><li>重复步骤3.7至3.9，直到没有更多的细胞可以脱落合理的肝晃动。 </li></ol> 4。肝细胞和SEC的净化<ol><li>离心机50毫升含3分钟，在150 XG细胞conicals。颗粒的肝细胞。 </li><li>转移包含非实质细胞，新鲜的50毫升冰conicals的液体部分。 </li><li>洗肝resuspending在缓冲区3沉淀，并重复步骤4.1和4.2三次。 </li><li>洗末，颗粒≥97％纯肝细胞。为了获得SECS，离心管C的所有ontaining池上清液在10分钟的200 XG（含造血干细胞，秒，以及KCS和一些小肝）。在4 ° C </li><li>吸缓冲区和4颗粒重新悬浮于5 mL的RPMI / BSA ° C。 </li><li>池的细胞聚集成2管，并添加35毫升的体积的RPMI / BSA的最多。 </li><li>离心3分钟，在100 XG conicals。 </li><li>收集前25毫升的液体转移到一个干净的50 mL锥形。 </li><li>重悬沉淀在余下的10毫升媒体，并添加25毫升冷的RPMI / BSA和离心3分钟，在100 XG。 </li><li>再次重复步骤3.8和3.9，弃细胞沉淀和低10毫升媒体。 </li><li>离心supernatents颗粒秒，KCS，造血干细胞在10分钟200 XG。在4 ° C </li><li>准备三个50毫升20毫升25％Percoll上冰的PBS管。底图与15毫升50％，每管Percoll。 </li><li>在总体积为30 mL的RPMI / BSA重悬细胞沉淀。 </li><li>仔细叠加选管会10毫升的细胞和离心20分钟，在900 XG h梯度在4 ° C </li><li> SECS和KCS有着非常密切的浮力密度和25/50％界面。造血干细胞通常在25％/媒体接口由于其较高的脂质储存细胞的浮力。任何剩余的肝细胞和血细胞降低buoyancies颗粒。吸从25/50％界面附近的自上而下和丢弃这种材料。 </li><li> 25/50％接口转移到与冷的RPMI新鲜锥形收集细胞。最终体积应〜40毫升，以稀释的Percoll。 </li><li>锥形离心10分钟350 XG在4 ° C沉淀细胞。 </li><li> 〜10毫升预热R​​PMI酸洗玻璃培养皿中含有100 U /毫升Penicillin/100克/ ml链霉素和地方细胞或结晶盘重悬细胞。 </li><li>孵育15分钟的细胞。在37 ° C，在一个组织文化的孵化器。 </li><li>岩石或旋流板一点点然后收集上清SECS。 KCS坚持比秒更迅速地在玻璃上。此外，SECS可从KCS分隔，使用抗CD31或anti-Stabilin2/HARE抗体结合磁珠immunopurification。 </li><li>台盼蓝排除使用hemacytometer或使用自动细胞计数器的生存能力和细胞数量可能被评定。 </li><li>文化的SECS纤维连接蛋白涂层的塑料菜在37 ° C，5％CO 2，与血管内皮生长因子为40毫微克/毫升，100 U / ml青霉素，100μg/ mL链霉素或牛血清白蛋白与肝细胞条件培养基RPMI/0.25 ％。 </li><li>肝细胞，KCS和SECS标记材料的内化的可评估使用伽玛计数器和规范使用这些培养方法细胞总蛋白或显微镜（荧光标记）。 </li></ol> 5。代表性的成果： 肝细胞净化≥97％和证券交易委员会的净化是表型盟友≥95％，使用这种方法。腹腔，门静脉插管，热烫的肝切除，都应该出现在一分钟内获得最佳效果。 HARE/Stabilin-2是对肝脏SECS的特异性受体，并不表示在其他类型的肝细胞。在此示例中，肝和SECS细胞裂解物5％的SDS - PAGE分离，并用单克隆抗体检测对Stabilin - 2两种异构体（ 图3） 。 普通肝素注入血液流是由肝脏清除16。间隙是主要由肝脏SECS肝细胞和KCS 17 。 Stabilin-2/HARE受体是主要清除受体结合和内化秒18的肝素。在我们的例子中，我们标记羧酸葡萄糖/ NS组与生物素标记的肝素。碘化链亲和素生物素是共轭化的肝素PROT检测eoglycan。 ，随后由放血标记的肝素注射，注射后30分钟，让我们监察机关内在这种形式的肝素。在这短短的时间间隔，肝的主要通关器官（ 图4）。 为了更清楚地了解细胞类型是负责清除，我们增加了125 I - SA - B - HEP辅以0.05％BSA的RPMI媒体，并允许它通过肝脏循环20分钟。胶原酶消化和蜂窝净化，绝大多数标记肝素内肝细胞（ 图5）SECS。 <img src="/files/ftp_upload/3138/3138fig1.jpg" alt="图1"/> 图1基本的肝架构。 SECS的血窦形成的墙壁通常是孔（小群各界）。星状细胞（HSC）被发现在Disse间隙库普弗彗星英语学习者（KCS），它们通常存在于血窦微血管。肝Disse间隙中占有的另​​一边。 <img src="/files/ftp_upload/3138/3138fig2.jpg" alt="图2"/> 图2。灌注装置的示意图。一）设置设备在冲洗/洗肝血窦。 TBS是在1升容量瓶中。二）一个125〜60 ml缓冲液2 mL容量瓶中含有胶原酶用于消化肝脏，在一个封闭的电路。污水线也发生了变化，从废弃物容器，烧瓶乙通过削减橡胶瓶塞的缺口。氧行不浸入含胶原酶（乙烧瓶）的缓冲区，以避免起泡。在每个烧瓶塞子​​的缺口，让污水线的有效转移，以防止氧气的压力增加。 <img src="/files/ftp_upload/3138/3138fig3.jpg" alt="图3"/> 图3。 </s仲&gt;肝细胞的准备工作是不污染秒。从肝细胞的纯化批次（泳道1）和秒（巷2）细胞裂解液等量的5％的SDS - PAGE分离，涂抹到硝酸纤维素。单克隆抗体＃30这是对HARE/Stabilin-2两种异构体（315 kDa和190 kDa的）具体是用来探测的裂解物。 <img src="/files/ftp_upload/3138/3138fig4.jpg" alt="图4"/> 图4。标记在机关肝素分布。大鼠经尾静脉注射125 I - SA - B - HEP等待30分钟，然后exsanguinated。采血，以便不给任何机关虚高读数。 （CPM）的每一个器官，每分钟的计数正常化对器官的重量以克。 <img src="/files/ftp_upload/3138/3138fig5.jpg" alt="图5"/> 图5：在肝和SECS标记的肝素量。转速我媒体载有125余SA - B - HEP被允许通过一个完整的肝胶原酶消化和蜂窝净化20分钟分发。等量的肝细胞和SEC细胞蛋白裂解物进行了量化与Bradford法和伽玛计数器计数。 <img src="/files/ftp_upload/3138/3138fig6.jpg" alt="图6"/> 图6。Kupffer细胞分离SECS粘附在玻璃上。细胞被放置在一个酸洗玻璃结晶菜，并坚持15分钟在37℃，5％的CO 2 。含有非坚持细胞的上清轻轻众说纷纭，放在离心管。从玻璃上都坚持细胞（泳道1）非坚持细胞（泳道2）8％的SDS - PAGE分离裂解液，涂抹，并与抗体对CD163 Kupffer细胞的具体探讨。 <img src="/files/ftp_upload/3138/3138fig7.jpg" alt="图7"/> 纤维连接蛋白涂层的塑料上镀图 7秒6的菜肴孵育过夜，0.1％BSA的RPMI补充。 （一）100X（二）200X放大倍率相位对比图像。

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Liver Physiol. 294, 520-528 (2008).</li><li>Harris, E. N., Weigel, J. A., Weigel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+hyaluronan+receptor+for+endocytosis+(HARE/Stabilin-2)+is+a+systemic+clearance+receptor+for+heparin.">The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin.</a> J. Biol. Chem. 283, 17341-17350 (2008).</li><li>Knook, D. L., 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=Separation+of+Kupffer+and+endothelial+cells+of+the+rat+liver+by+centrifugal+elutriation.">Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation.</a> Exp. Cell. Res. 99, 444-449 (1976).</li><li>Graupera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+endothelial+COX-1-derived+prostanoids+modulate+the+hepatic+vascular+tone+of+cirrhotic+rat+livers.">Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 288, 763-770 (2005).</li><li>Yannariello-Brown, J., Zhou, B., Ritchie, D., Oka, J. A., Weigel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ligand+blot+assay+detects+different+hyaluronan-binding+proteins+in+rat+liver+hepatocytes+and+sinusoidal+endothelial+cells.">A novel ligand blot assay detects different hyaluronan-binding proteins in rat liver hepatocytes and sinusoidal endothelial cells.</a> Biochem. Biophys. Res. Commun. 218, 314-319 (1996).</li><li>Duryee, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+is+a+cofactor+for+malondialdehyde-acetaldehyde+adduct-mediated+cytokine/chemokine+release+by+rat+sinusoidal+liver+endothelial+and+Kupffer+cells.">Lipopolysaccharide is a cofactor for malondialdehyde-acetaldehyde adduct-mediated cytokine/chemokine release by rat sinusoidal liver endothelial and Kupffer cells.</a> Alcohol Clin. Exp. Res. 28, 1931-1938 (2004).</li></ol>

没有利益冲突的声明。

麻醉大鼠悬滴法在异氟醚蒸气诱导昏迷，是我们研究的首选方法。一个蒸发器，也可能被用来代替注射器与棉花维持在会议厅外的动物，但手术过程中的那么快，它不是必需的。聚乙二醇添加到异氟醚，以减少蒸汽压力和蒸发速度。异氟醚蒸气太多太快会诱发死亡。如果动物死亡前肝脏是空心的，用TBS，血液可能汇集在肝脏血块烫一下，防止血窦和胶原酶消化高效清洗。肝素除可作为抗凝剂，但不是在这些研究中使用，因为我们用我们的探头标记的肝素。 Gey的缓冲盐溶液（GBSS）往往是取代其他实验室秒净化。虽然它是非常有用的SEC的净化，HEPatocytes不容忍GBSS以及缓冲器1-3和存活率都差不多高。当测量的内吞作用，这是很好的做法，措施，在机关和纯化的肝细胞的背景水平。 这种方法的二分之一以下胶原酶肝脏灌注SECS高效净化。另一种方法是由淘洗离心19 Percoll梯度分离非实质细胞。使用超过Percoll梯度淘洗的优点是稍高的细胞存活率和数字。的缺点是，淘洗离心需要一个专门的转子，专用离心机，蠕动泵成本几万块钱。这种方法只需要使用冷藏表离心机，蠕动泵，这是许多实验室的标准。 SECS和选择性粘附KCS的分离是一种标准的方法20日至22日。秒，将坚持以玻璃和塑料虽然这是事实，关键的一点是使用不超过15分钟的孵化酸冲洗干净玻璃，在37 ° C时，5％的CO 2。 KCS将始终坚持比秒的速度更快，最好是轻轻用媒体的KCS提取任何剩余的，已成为松散连接SECS。链霉蛋白酶和其他蛋白酶也省略了在本议定书中的胶原酶消化步骤，增加细胞的存活率。蛋白酶消化细胞外受体，并可能抑制细胞粘附在最后的纯化步骤。 这里介绍的方法具有广泛的应用。虽然我们显示肝素初步过关，获得原代肝细胞肝脏灌注的最终结果，KC，秒，造血干细胞可能是有用的研究，涉及的代谢途径，免疫调节，清道夫活动，及其他数生理studiES。在此过程中最困难的部分是良好的插管，消化，肝脏和肝脏的处理，而无需来自门静脉的导管滑。

<ul><li>缓冲区1：氯化钠6.7毫米氯化钾，142毫米10毫米的HEPES，1.5％BSA，pH值7.4 </li><li>缓冲区2（灌注缓冲区）：67.0毫米氯化钠，6.7 mM的氯化钾，4.8毫米氯化钙2 - H2O，101.0 mM的HEPES，BSA 1.5％，pH值7.4。 </li><li>缓冲区3：137毫米氯化钠，氯化钾4.7毫米，0.7毫米硫酸镁4，1.2毫米氯化钙2 · 2H 2 O，10毫米的HEPES，1.5％BSA，pH值7.4 </li><li> RPMI / BSA：15克/ L，BSA的RPMI </li><li> TRIS缓冲液（TBS）：154 mM氯化钠，10毫米Trizma基盐。 </li><li>胶原酶：胶原酶一个罗氏（＃11088785103），沃辛顿生化或胶原酶我的胶原酶I型（＃LS004691）（＃C0130）或IA（＃C9891）自Sigma - Aldrich。 </li></ul>所有盐自Sigma - Aldrich <table border="1"><tbody><tr><td>名称</td><td>公司</td><td>型号/目录＃ </td><td>评论</td></tr><tr><td> 20L水浴</td><td> ThermoFisher </td><td> 2231 </td><td>水是约45 ° C，以弥补冷却</td></tr><tr><td></td><td></td><td></td><td>管内。 </td></tr><tr><td>蠕动泵</td><td>科尔 - 帕默</td><td> 7553-70 </td><td>的Masterflex系列</td></tr><tr><td>冷冻离心机</td><td> Sorvall </td><td>联想XTR </td><td></td></tr><tr><td>导管</td><td> BD公司</td><td> 381444 </td><td></td></tr><tr><td> Dessicator室</td><td>费舍尔</td><td> 08595E </td><td>使用内部板</td></tr><tr><td> Percoll </td><td>西格玛</td><td> P4937 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> SeraCare </td><td> AP - 4510 - 01 </td><td></td></tr><tr><td> 100微米网状</td><td>光谱实验室</td><td> 146488 </td><td></td></tr><tr><td> 30微米网状</td><td>光谱实验室</td><td> 146506 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 21870 </td><td></td></tr><tr><td>聚乙二醇</td><td>光谱实验室小号</td><td> PO107 </td><td></td></tr></tbody></table>

in vivo liver endocytosis followed by purification of liver cells by liver perfusion

肝脏是哺乳动物体内的代谢中心，并作为血液过滤器。肝脏的基本架构说明图1中，其中超过85％的肝脏肿块是由肝细胞和其余15％的细胞质量是枯否细胞（KCS），星状细胞（HSC）组成，正弦内皮细胞（秒）。 SECS形成肝脏内的血管壁，称为窗孔在细胞质内含有专门的形态。细胞质开窗孔的外观（〜100微米）的细胞内秒的行为，使筛子其中最乳糜微粒，乳糜微粒残余物和大分子，但不是细胞，通过向肝细胞和造血干细胞 1（图1）。由于缺乏基底膜，SECS和肝细胞之间的差距Disse间隙。造血干细胞占据这个空间，并发挥了突出的作用，在调控和应对我njury，贮存维甲酸和免疫调节的肝2。 SECS是身体endocytically最积极的显示阵列上其细胞表面的 3的清道夫受体细胞。这些措施包括Stabilin SR - A - 1和Stabilin - 2。一般来说，小的胶体颗粒小于230纳米的大分子缓冲阶段秒，而，大颗粒和吞噬细胞碎片是由KCS 4（吞噬） 。因此，如从血液中糖胺外物质的散装间隙很大程度上取决于对健康和 5,6秒内吞功能。例如，增加血液中的透明质酸水平是指示性的肝脏疾病，从轻微到更严重的形式 7 。 随着一个报告8外，还有SEC存在没有永生细胞株。这永生细胞株，即使是去分化ated，它不表达清道夫受体（我们的数据，未显示）的主要SECS。所有细胞生物学的研究，必须从动物新鲜获得的原代细胞。不幸的是，SECS去分化标准培养条件下，必须在1或2天从动物隔离后使用。 Stabilin - 2的表达或野兔受体 9，CD31，存在细胞质开窗 1秒分化标记。除了 ​​血管内皮生长因子的培养基，或在肝细胞条件培养基培养10,11细胞，可以延长秒分化。 在这份报告中，我们将展示完整的器官内吞活动的SECS使用无线电标记的肝素，透明质酸为美国证券交易委员会的具体Stabilin - 2受体。然后，我们将净化灌注肝脏的肝细胞和SECS来衡量的内吞作用。

Watch this Scientific Journal Video about In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion at JoVE.com

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

从动物获得的无细胞系存在的主要细胞，肝窦内皮细胞（秒）的研究必须执行。这种方法依赖于肝的消化和差速离心SEC随后的培养和实验净化。

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

in-vivo-liver-endocytosis-followed-purification-liver-cells-liver

Research

JoVE Journal

Biology

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

19.0K 次观看.  University of Nebraska, Lincoln. 从动物获得的无细胞系存在的主要细胞，肝窦内皮细胞（秒）的研究必须执行。这种方法依赖于肝的消化和差速离心SEC随后的培养和实验净化。

视频: In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

科研

生物化学

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

组织细胞外囊泡与肝脏分离

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

生物学

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. ...

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

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

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 ...

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 and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

小鼠移植肝球体的无创眼内活体成像

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

作者聚焦：利用小鼠眼腔进行无创肝球研究

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

移植到小鼠眼前房的肝球体的体内成像

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

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此视频中的章节

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

组织细胞外囊泡与肝脏分离

大鼠门户成纤维细胞的分离

人肝细胞通过一个两步胶原酶灌注过程隔离

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

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

多参数灌注对照分离原代大鼠肝细胞

小鼠原发性肝正弦内皮细胞的分离与表征

移植到小鼠眼前房的肝球体的体内成像

移植到小鼠眼前房的肝球体的体内成像