提供资金的部分由 NIH 从赠款 R01HL130864。

本议定书所概述的所有动物程序都已由林肯-内布拉斯加州大学的机构动物照料和使用委员会 (IACUC) 根据《议定书》 #1435 批准。
1. 准备
<ol><li>解决方案和培养培养基的制备<ol>
<li>根据材料表准备所有区域性媒体和缓冲区。</li>
	</ol>
</li>
	<li>仪器的准备<ol>
<li>在42摄氏度设置一个水浴 (> 10 升), 一个可变速度的蠕动泵, 和保存烧瓶的夹子。准备弯剪刀和镊子 (图 1)。</li>
		<li>在水浴上或旁边, 设置一个烘烤板 (约 38 x 26 厘米) 与发泡胶垫 (50 毫升圆锥机架), 覆盖与吸收垫切割成 20 x 20 厘米片 (图 1)。</li>
		<li>在附近放置一块屏蔽胶带 (5-8 厘米)。放置一个20厘米涤纶缝纫线附近, 是削减和准备使用。获得一个可伸缩针的塑料导管 (7 毫米 x 19 毫米,图 1)。</li>
	</ol>
</li>
	<li>玻璃器皿的制备<ol>
<li>使用夹子, 放置和固定在水浴中的1升烧瓶, 其中包含200毫升 1x PBS 与一个塞子, 有两个1毫升吸管进入解决方案。橡胶软木有缺口, 允许循环液体在一个封闭的电路。</li>
		<li>与另一个钳位, 放置一个125毫升瓶包含45毫升的缓冲2连同一个塞子, 有两个1毫升吸管, 其中之一是在液体附近的瓶子和其他保持在液体和吹氧到烧瓶。 注: 在塞子的每个吸管将有一个快速断开适配器连接到年底, 以方便切换油管。氧气将通过在塞子中不浸入液体中的吸管轻轻地流入两个烧瓶中。</li>
		<li>留出两个不育的结晶皿。</li>
	</ol>
</li>
</ol>2. 动物做法
<ol><li>预热 pbs 和缓冲2解决方案到42°c 在水浴和流通 PBS 至少20毫升/分钟在管道中的封闭电路, 以保持液体线温暖。浸入水浴中的任何多余油管保持尽可能接近42摄氏度。将油管的男性端排入含有 PBS 的1升烧瓶中。</li>
	<li>将一个棉球放在吸水垫上, 并添加约2毫升30% 异氟醚 (由聚乙烯乙二醇200组成, 通过转移吸管降低了异氟醚的蒸发率)。</li>
	<li>拿起鼠标的尾巴, 把它放在一个结晶的菜肴, 然后迅速翻转到棉花球的菜, 使鼠标有一个小的空间, 其中吸入麻醉。观察鼠标的呼吸速度, 确保鼠标在麻醉下有效。 注: 呼吸速率应较慢和更深, 尾部松弛, 爪子响应麻醉下的捏试验;有关其他详细信息, 请参阅 IACUC 指南。</li>
	<li>当鼠标在麻醉下 (这需要1-2 分钟), 准备一个10毫升注射器的桶, 拔出活塞和插入一个小棉球。将30% 异氟醚的一毫升, 用转移吸管添加到枪管内的棉球上, 并将枪管放在桌子上, 等待老鼠变得不省人事。</li>
	<li>快速摘下结晶盘, 翻转鼠标背部, 把注射器桶放在鼻子上。双检查呼吸率, 脚趾捏, 和松弛的尾巴。任何对脚趾捏和松弛的尾巴的反应表明, 老鼠没有完全在麻醉下。把注射器桶放在鼻子上保持无意识。</li>
	<li>把拇指钉通过鼠标的爪子, 四肢伸出仰卧位。用70% 异丙醇或乙醇将腹部和肋骨保持湿润。</li>
	<li>用直钳一只手, 抬起靠近腹部底部的皮肤。另一方面用剪刀剪帐篷的皮肤和腹膜。切口应该是水平的或在帐篷皮肤的底部。一定要切开所有层的皮肤和腹膜, 以获得进入肠道。侧面切开在腹部附近由肋骨笼子在两边没有偷任何器官。</li>
	<li>通过下肋笼切开皮瓣。用镊子的背部将肠道向右移动, 以暴露门静脉。 注: 动物出血应尽量少;该动物仍应呼吸和深度麻醉。</li>
	<li>将闭合的弯曲钳放置在肝与上级十二指肠静脉之间的门静脉 (图 3箭头)。</li>
	<li>在门静脉下方打开镊子。抓住线程, 仔细拉它通过, 使它在门静脉的中心。在门静脉周围系上一个上手结, 而不紧。</li>
	<li>将弯曲钳放在门静脉下, 轻轻地将其拉向鼠标尾部, 以理顺静脉 (图 4A)。</li>
	<li>另一方面, 用导管, 将针面的斜角与镊子附近的门静脉下部平行 (图 4B)。</li>
	<li>用针轻轻穿刺静脉 (图 4C)。确保针的斜角在静脉腔内。收回弹簧针, 继续通过静脉推入聚合物导管, 直到斜面靠近静脉分支区域。这是肝脏内的如果导管正确放置, 血液的回流就会可见。(图 4D)。</li>
	<li>拧紧的上手结和拉它在导管, 以帮助稳定它。立即将泵浦率从20毫升/分钟降低到4毫升/分, 并切开另一条主要的血管进行引流。切除主动脉腹型紫癜以获得最佳结果。</li>
	<li>将油管的雄性末端置于导管的母端 (图 5A)。确保生产线上没有气泡。小心, 导管不是被推入肝脏或静脉。线程的放置并不重要, 但它有助于防止从静脉回流, 并有助于保持导管在正确的位置 (图 5B, C)。</li>
	<li>使用屏蔽胶带将油管固定在垫上。使用直钳挤压出水血管, 使肝脏膨胀几次, 以确保血液耗尽 (图 5D)。 注: 标准实验室胶带可能无法正常工作;然而, 即使在潮湿的情况下, 屏蔽胶带仍会粘在垫上。</li>
</ol>3. 肝脏灌注
<ol><li>当肝脏冲洗与 PBS, 测量出约24毫克胶原酶 IV 型, 并把它放在含有45毫升缓冲2在水浴瓶。一定要旋流的液体在瓶中, 使胶原酶完全溶解, 并把瓶子放回钳, 使含有部分的液体瓶被完全淹没在水中。</li>
	<li>观察肝脏的颜色变化, 因为它是由 PBS 冲洗。将流出油管从1升烧瓶改为125毫升烧瓶。不要让气泡流入肝脏。</li>
	<li>一旦灌注缓冲到达肝脏, 简单地挤压排出的流出血管, 在血管内积聚一些压力, 让这个液体填满肝脏的所有裂片。确保不要把引流时间过长, 因为这可能会爆裂肝脏周围的薄结缔组织 (Glisson 的胶囊), 并破坏肝脏毛细血管床内的液体灌注或流动。</li>
	<li>允许缓冲2与胶原酶灌注通过肝脏, 直到所有45毫升已经流经组织。 注: 如果成功, Glisson 的胶囊应分离的实质或肝脏组织和肝脏本身应该出现无定形。</li>
	<li>增加约10毫升的缓冲1到其他结晶皿和放置在鼠标旁边。</li>
	<li>取出导管并关闭泵。</li>
	<li>用直钳和剪刀, 从老鼠身上切下肝脏。如果胶原酶消化非常有效, 可能需要有一个干净的, 不育的勺子手从老鼠舀肝, 并把它放在缓冲1。</li>
</ol>4. 肝细胞纯化
注意: 在无菌组织培养罩中进行细胞的操纵, 以限制细胞在所有后续步骤中的培养。
<ol><li>采用无菌技术, 用镊子抓肝, 轻轻摇动肝细胞。撕裂或拉回 Glisson 的胶囊: 当细胞从肝脏中摇动时, 缓冲会变得不透明。</li>
	<li>将细胞溶液倒入50毫升圆锥, 用100µm 过滤器放置在顶部。</li>
	<li>增加更多的缓冲1到肝脏残余和继续震动细胞。继续, 直到肝脏似乎没有细胞, 或当未消化的部分肝脏不再产生细胞。</li>
	<li>将细胞液倒入另一50毫升圆锥, 其中含有40µm 过滤器。</li>
	<li>离心机圆锥在摆动斗转子在 100 x g 3 分钟。 注: 不要使用最大刹车, 因为这可能会去除细胞颗粒。使用刹车在 80%, 这是足够的所有剩余的离心步骤在4°c</li>
	<li>倒出上清 (含有 npc 和死肝细胞) 成一个干净的圆锥状, 并把它放在冰上。确保这是在一个议案, 以观察颗粒附着。</li>
	<li>并用重悬40毫升的缓冲液中的颗粒1。重复步骤4.5 和4.6。并用重悬40毫升的缓冲液中的颗粒3。重复步骤4.5 和4.6 两次。</li>
	<li>并用重悬的纯化肝细胞在温暖的 DMEM + 10% 血清 + 2x 青霉素-链霉素 (笔/链球菌)。</li>
	<li>如果细胞被培养在胶原涂层板上, 允许他们坚持1小时, 并更换培养基至少一次, 因为这将防止任何新生的细菌污染。<ol>
<li>用0.05% 胶原蛋白在0.001% 醋酸中孵化出胶原膜, 至少1小时37摄氏度, 然后用 1x PBS 进行洗涤。</li>
	</ol>
</li>
	<li>如果肝细胞生存能力低, 并且渴望获得纯化的高活性肝细胞, 那么请使用从克瑞默et al10中适应的以下协议。<ol>
<li>混合9卷 pvp 溶液 (Percoll, 23% 瓦特/w 溶液的水和 15-30 纳米胶体二氧化硅粒子涂在 polyvinylpyrrolidone 的密度离心) 和1体积 10x HBSS, 使 iso 渗透 PVP 解决方案 (SIP)。</li>
		<li>将24毫升的 SIP 添加到一个不育的50毫升圆锥锥, 可储存在这些条件下长达2月, 在4摄氏度。</li>
		<li>将肝细胞浓度调整为 5-10 x 106细胞/mL, 其培养基如步骤4.8 所述。</li>
		<li>将25毫升的细胞悬浮液添加到每24毫升含圆锥的 SIP 中, 并通过柔和的反转进行混合。这个溶液的密度是1.06 克/毫升。</li>
		<li>离心机圆锥在 50 x g 10 分钟在4°c。</li>
		<li>吸入 SIP 和絮凝剂, 并用重悬细胞 HBSS。</li>
		<li>通过离心 50 x g 10 分钟, 在4摄氏度清洗细胞。</li>
		<li>重复步骤4.10.6 再一次, 然后并用重悬细胞生长培养基。</li>
	</ol>
</li>
</ol>5. SEC 净化
<ol><li>通过旋转管子在 163 x g 为10分钟, 将上清液中的细胞从步骤4.6 中颗粒化. 将上清液从试管中丢弃, 并并用重悬在5毫升 RPMI 中的所有细胞小球, 不带血清, 并将它们聚集在一根管子中。</li>
	<li>一旦所有的颗粒被汇集, 添加 RPMI 到最终的体积35毫升。</li>
	<li>离心机的池锥在 25 x g 3 分钟小心地吸入前25毫升的 RPMI 与25毫升吸管, 并放置此媒体包含 npc 在一个干净的50毫升锥形冰。</li>
	<li>加入25毫升新鲜 RPMI 回管, 并用重悬颗粒。</li>
	<li>重复步骤5.3 第二次清洗和池中清。丢弃剩余的10毫升介质和颗粒 (颗粒主要由死肝细胞组成)。将池上清液离心机 163 x 克, 10 分钟。</li>
	<li>在离心过程中, 在50毫升圆锥内准备 PVP 溶液梯度。</li>
	<li>添加15毫升 50% PVP 溶液的50毫升圆锥后跟20毫升 25% Percoll 覆盖 pipettor 设置为最慢的弹射速度。一定要观察界定两层的15毫升标记上的折射线, 因为这是秒和 KCs 在离心后会聚集的地方。</li>
	<li>并用重悬在10毫升的冷 RPMI 中, 先前在步骤5.5 中颗粒化的细胞, 并将它们覆盖到 PVP 溶液梯度上。确保在两层之间保持明确的分界。离心机的梯度在 805 x g 20 分钟与刹车设置在50%。</li>
	<li>从上向下的方向吸入到管子上的20毫升标记上, 然后丢弃这种材料。使用5毫升转移吸管, 收集位于 25/50% 接口上的秒和 KCs。丢弃任何褐色的细胞簇, 因为这些都是死肝细胞。</li>
	<li>将细胞放置在50毫升锥形中, 并添加 RPMI 至50毫升标记, 以稀释 PVP 溶液。离心机在 200 x g 10 分钟, 80% 刹车。</li>
	<li>在12毫升的温暖 RPMI 中, 吸入并丢弃上清和并用重悬颗粒, 同时洗涤圆锥锥。</li>
	<li>将上清放入聚苯乙烯培养皿中, 并在室温下在湿润的组织培养孵化器中孵化8分钟, 将该秒从 KCs 中分离出来。</li>
	<li>用25毫升的吸管吸进介质, 用相同的介质冲洗盘子, 以最低的速度 pipettor 设置收集剩余的秒, 而 KCs 坚持板。</li>
	<li>离心机秒在 200 x g 10 分钟和并用重悬在 RPMI + 5% 血清 + 笔/链球菌。然后滴下细胞, 并放置在胶原蛋白涂层的文化菜肴。</li>
</ol>

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J Cell Biol. 43 (3), 506-520 (1969).</li><li>Seglen, P. O. <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+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Miller, L. L., Bly, C. G., Watson, M. L., Bale, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dominant+role+of+the+liver+in+plasma+protein+synthesis;+a+direct+study+of+the+isolated+perfused+rat+liver+with+the+aid+of+lysine-epsilon-C14.">The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14.</a> J Exp Med. 94 (5), 431-453 (1951).</li><li>Edstrom, S., Ekman, L., Ternell, M., Lundholm, K. <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+mouse+liver+cells:+perfusion+technique+and+metabolic+evaluation.">Isolation of mouse liver cells: perfusion technique and metabolic evaluation.</a> Eur Surg Res. 15 (2), 97-102 (1983).</li><li>Meyer, J., Gonelle-Gispert, C., Morel, P., Buhler, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Isolation+and+Purification+of+Murine+Liver+Sinusoidal+Endothelial+Cells:+A+Systematic+Review.">Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review.</a> PLoS One. 11 (3), 0151945 (2016).</li><li>Sies, 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+use+of+perfusion+of+liver+and+other+organs+for+the+study+of+microsomal+electron-transport+and+cytochrome+P-450+systems.">The use of perfusion of liver and other organs for the study of microsomal electron-transport and cytochrome P-450 systems.</a> Methods Enzymol. 52, 48-59 (1978).</li><li>Smedsrod, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+preparation+of+mouse+liver+Kupffer+cells+and+liver+sinusoidal+endothelial+cells.">Protocol for preparation of mouse liver Kupffer cells and liver sinusoidal endothelial cells.</a> Munin open research archive. , 1-10 (2012).</li><li>Smedsrod, B., Pertoft, H., Eggertsen, G., Sundstrom, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+characterization+of+cultures+of+Kupffer+cells+and+liver+endothelial+cells+prepared+by+means+of+density+separation+in+Percoll,+and+selective+substrate+adherence.">Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence.</a> Cell Tissue Res. 241 (3), 639-649 (1985).</li><li>Meyer, J., Lacotte, S., Morel, P., Gonelle-Gispert, C., Buhler, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+mouse+liver+sinusoidal+endothelial+cell+isolation.">An optimized method for mouse liver sinusoidal endothelial cell isolation.</a> Exp Cell Res. 349 (2), 291-301 (2016).</li><li>Kreamer, B. 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=Use+of+a+low-speed,+iso-density+percoll+centrifugation+method+to+increase+the+viability+of+isolated+rat+hepatocyte+preparations.">Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.</a> In Vitro Cell Dev Biol. 22 (4), 201-211 (1986).</li><li>Meijer, D. K., Keulemans, K., Mulder, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+perfused+rat+liver+technique.">Isolated perfused rat liver technique.</a> Methods Enzymol. 77, 81-94 (1981).</li><li>Gopalakrishnan, S., Harris, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+liver+endocytosis+followed+by+purification+of+liver+cells+by+liver+perfusion.">In vivo liver endocytosis followed by purification of liver cells by liver perfusion.</a> J Vis Exp. (57), e3138 (2011).</li><li>Cook, 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=.">.</a> The Anatomy of the Laboratory Mouse. , (1965).</li><li>Liu, J., 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=Advanced+Method+for+Isolation+of+Mouse+Hepatocytes+Liver+Sinusoidal+Endothelial+Cells,+and+Kupffer+Cells.">Advanced Method for Isolation of Mouse Hepatocytes Liver Sinusoidal Endothelial Cells, and Kupffer Cells.</a> Methods Mol Biol. 1540, 249-258 (2017).</li><li>Knobeloch, D. E., Ehnert, S., Schyschka, L., Buchler, P., Schoenberg, M., Kleeff, J., Thasler, W. E., Nussler, N. C., Godoy, P., Hengstler, J., Nussler, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Hepatocytes:+Isolation,+Culture,+and+Quality+Procedures.">Human Hepatocytes: Isolation, Culture, and Quality Procedures.</a> Methods in Molecular Biology. 806, 99-120 (2012).</li><li>Clarke, B. L., 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=Recycling+of+the+asialoglycoprotein+receptor+in+isolated+rat+hepatocytes.+ATP+depletion+blocks+receptor+recycling+but+not+a+single+round+of+endocytosis.">Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis.</a> J Biol Chem. 260 (1), 128-133 (1985).</li></ol>

作者没有什么可透露的。

此协议突出显示了可用的工具, 这将使用户在该过程中获得很高的成功率。在使用主单元格时, 成功的灌注是任何下游应用程序的基础。
确定成功的过程有几个关键步骤。首先, 导管提示放置在门静脉内必须是正确的。如果放置在肝脏太远, 只有小裂片将被灌注。提示应位于门静脉的右、左分支下方。在针上使用高分子复合导管的优点是, 针的倒钩比导管在灌注过程中更容易撕裂静脉。其次, 胶原酶的质量和数量决定了肝脏的消化效率。在这个过程中, 使用了预合格的胶原酶4型, 它是悬浮在0.5 毫克/毫升的缓冲 2, 如果要测定的胶原酶活性大于900。
在胶原酶灌注的最后, 肝脏应该保留一个有点暗褐色到褐色的颜色。如果它是浅褐色的颜色, 那么大部分的肝细胞都死了。当从老鼠身上剪下来时, 肝脏就会崩溃。在最好的条件下, 肝脏可以用小勺子从老鼠的体腔里挖出来。肝脏在这种情况下总是给予非常高的细胞活力。如果要花很大力气把细胞分开, 如果肝脏在萃取过程中仍然牢固, 或者如果有大量的力量需要通过过滤器移动肝细胞, 那么肝细胞的生存能力就会降低。肝细胞用微绒毛覆盖, 使它们具有很高的表面面积 (图 10)。不完全消化会保留肝细胞之间的胞细胞连接, 而机械剪切则会撕裂细胞膜。原位灌注胶原酶是分离细胞和维持高生存能力的最佳方法。在图 11中, 进行了两种肝细胞制剂, 在缓冲1中进行少量洗涤后, 对细胞颗粒进行比较。左侧的颗粒较轻, 其细胞活力约为50%。右侧的颗粒较暗, 其生存能力为92%。类似地, 当液体从50毫升锥形中倒入时, 深色颗粒在管内是固定的, 与打火机颗粒形成对比, 在液体被浇掉时, 它会在管子中滑动。当肝脏有高硬化程度时, 可能发生低细胞活力或低效显影。
由于活肝细胞的密度高于死肝细胞, 离心过程将导致在可行的肝细胞中高度纯净的准备工作15。如果有大量的死亡肝细胞 (通常发生在条件是次优), 活细胞可以进一步丰富, 并与死细胞分离使用 PVP 梯度。此外, 由于活肝细胞颗粒比死肝细胞快, 3 号球团的顶部rd的吸入也会增加颗粒中活细胞与死体细胞的比值。这些步骤是快速简便的方法, 在需要时将活体与死肝细胞和细胞碎片分开,10,16。这是特别有用的, 如果样品是珍贵的, 只有数以百万计的细胞是必要的实验。
总之, 这是一个简单而有效的方法来收获肝细胞和秒。按目前的价格, 执行这一程序的成本, 包括所有试剂和一次性耗材是低于 75 USD 每准备。如果需要多只老鼠, 最好是进行肝细胞纯化, 并保持在冰上的 NPC 分数, 直到所有的老鼠被处理。npc 通常在冰上稳定至少5小时, 但更长的时间没有被测试在这个实验室。

胶原酶灌注肝脏获得肝细胞已执行自二十世纪五十年代代初, 并不断改善后,1,2,3,4。在迈耶et al5中, 对许多用于肝细胞纯化的方法学、技术和试剂进行了非常好的回顾。获得令人满意的高活性肝细胞产量和秒是技术上的挑战。分离细胞的机械力, 包括胶原酶的质量, 是一些难以控制的变量。由于肝细胞对机械力的敏感性, 在亚优条件下它们的生存能力明显降低, 这促使需要一个描述最佳隔离条件的协议。秒似乎不像对机械剪切敏感。原位灌注胶原酶消化是迄今为止最好的方法来破坏细胞连接, 以获得单细胞制剂从肝脏和其他器官, 如肠道和脾脏6。该协议演示了一种简单的方法, 通过使用可伸缩的塑料导管, 而不是切口, 可以导致门静脉崩溃, 在门静脉中引入灌注缓冲, 如 Smedsrød et al7中所述。
这篇手稿的目的是为了证明在肝脏丰富的程序中成功所需的最关键的步骤。这些步骤包括导管放置, 灌注液体的流动和消化后组织的处理。流速调节在血液流量的自然速率之上, 但足够低, 以保持 Glisson 的胶囊完好无损。一旦肝脏被正确地消化, 细胞在溶液中分离, 细胞纯化就比较简单, 无论是通过差异离心、板粘连还是通过磁珠提纯。活肝细胞密度较高, 易于从非实质细胞 (npc) 和慢速离心死肝细胞中纯化。对于大多数应用来说, 分离枯否细胞 (KCs) 和秒的板粘连是一种常见的方法8, 但有报告说它不产生秒9的最佳纯度。KCs 有倾向坚持坚实刚性表面迅速和培养皿 (标准聚苯乙烯) 是最常用的材料为这个做法。SEC 或 KC 使用抗体共轭磁性珠子进行纯化无疑是对这些细胞进行显著纯化的最佳方法, 尽管该程序在该协议上增加了 3-4 h, 但总产量减少了9。本报告详细说明了最佳肝脏灌注的过程, 通常产生大量可行细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >&#160;1 L Erlenmeyer flask</td>
			<td >Fisher Scientific</td>
			<td >S63274</td>
			<td ></td>
		</tr>
		<tr >
			<td >250 mL Erlenmeyer flask</td>
			<td >Fisher Scientific</td>
			<td >S63271</td>
			<td></td>
		</tr>
		<tr >
			<td >silicone tubing&#160;</td>
			<td >Cole-Parmer</td>
			<td >96400-14</td>
			<td>This tubing runs from the flasks through the pump to the T connector and then to the 1.0 mL syringe that is connected to the catheter.</td>
		</tr>
		<tr >
			<td >Tygon tubing</td>
			<td >Fisher Scientific</td>
			<td >R3603</td>
			<td>Used as an adaptor between 96400-14 and pipettes and T connector.&#160; This may also be used for the oxygen tubing.</td>
		</tr>
		<tr >
			<td >T-connectors</td>
			<td >Cole-Parmer</td>
			<td >EW-06294-82</td>
			<td></td>
		</tr>
		<tr >
			<td >Quick dissconnects</td>
			<td >Fisher Scientific</td>
			<td >6150-0010</td>
			<td></td>
		</tr>
		<tr >
			<td >Pinch Clamps</td>
			<td >Fisher Scientific</td>
			<td >6165-0002</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Variable speed Pump, model 7553-70</td>
			<td >Cole-Parmer</td>
			<td >EW-07559-00</td>
			<td>Periplastic pump with variable speed</td>
		</tr>
		<tr >
			<td >Pump head, model 7014-20</td>
			<td >Cole-Parmer</td>
			<td >EW-07014-20</td>
			<td></td>
		</tr>
		<tr >
			<td >glass graduated 1.0 mL pipettes&#160;</td>
			<td >Fisher Scientific</td>
			<td >13-678</td>
			<td></td>
		</tr>
		<tr >
			<td >curved non-serrated scissors</td>
			<td >Fine Science Tools</td>
			<td >14069-12</td>
			<td></td>
		</tr>
		<tr >
			<td >Dumont forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-20</td>
			<td></td>
		</tr>
		<tr >
			<td >Curved forceps</td>
			<td >Fine Science Tools</td>
			<td >13009-12</td>
			<td></td>
		</tr>
		<tr >
			<td >10 mL syringe</td>
			<td >Fisher Scientific</td>
			<td >03-377-23</td>
			<td>Only barrel of syringe will be needed</td>
		</tr>
		<tr >
			<td >sterlized spoon</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Cotton ball(s)</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Polyester sewing thread</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Masking tape</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >thumb tacks</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >styrofoam pad&#160;</td>
			<td >50 mL conical rack</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >cookie/baking sheet</td>
			<td >Home supply store</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Absorbant underpads</td>
			<td >Fisher Scientific</td>
			<td>14-206-64</td>
			<td></td>
		</tr>
		<tr >
			<td >19 L water bath</td>
			<td >Fisher Scientific</td>
			<td >TSCOL19</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Insyte Autogaurd Shielded IV Catheter 24 guage</td>
			<td >Becton Dickinson</td>
			<td >381412</td>
			<td>Plastic cathetar with retractable needle</td>
		</tr>
		<tr >
			<td >Crystallizing dishes 100x50</td>
			<td >VWR&#160;</td>
			<td >89000-290</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene petri dishes</td>
			<td >Sigma Aldrich</td>
			<td >P5481-500EA</td>
			<td></td>
		</tr>
		<tr >
			<td >50 mL conical tubes</td>
			<td >Fisher Scientific</td>
			<td >12-565-270</td>
			<td></td>
		</tr>
		<tr >
			<td >graduated pipettes (5 mL)</td>
			<td >Fisher Scientific</td>
			<td >170355</td>
			<td></td>
		</tr>
		<tr >
			<td >graduated pipettes (25 mL)</td>
			<td >Fisher Scientific</td>
			<td >170357</td>
			<td></td>
		</tr>
		<tr >
			<td >EasyStrainer 100 &#956;M</td>
			<td >Greiner bio-one</td>
			<td >542000</td>
			<td>100 &#956;m filter</td>
		</tr>
		<tr >
			<td >EasyStrainer 40 &#956;M</td>
			<td >Greiner bio-one</td>
			<td >542040</td>
			<td>40&#956;m filter</td>
		</tr>
		<tr >
			<td >Sterile transfer pipettes</td>
			<td >Fisher Scientific</td>
			<td >13-711-20</td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated swinging bucket centrifuge</td>
			<td >Sorvall Legend XTR</td>
			<td >75-217-406</td>
			<td>Centrifuge with swinging bucket rotar</td>
		</tr>
		<tr >
			<td >Galaxy 170R tissue culture incubator&#160;</td>
			<td >Eppendorf</td>
			<td >CO170R-120-0000</td>
			<td>Humidified tissue culture incubator</td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Compound</td>
			<td >Grams (g/L)</td>
			<td>Millimolar (mM)</td>
		</tr>
		<tr >
			<td >Buffer 1, pH 7.4</td>
			<td >NaCl</td>
			<td >8.3</td>
			<td >142</td>
		</tr>
		<tr >
			<td ></td>
			<td >KCl</td>
			<td >0.5</td>
			<td >6.7</td>
		</tr>
		<tr >
			<td ></td>
			<td >HEPES</td>
			<td >2.4</td>
			<td >10</td>
		</tr>
		<tr >
			<td ></td>
			<td >BSA</td>
			<td >15</td>
			<td >0.226</td>
		</tr>
		<tr >
			<td >Buffer 2, pH 7.4</td>
			<td >NaCl</td>
			<td >3.9</td>
			<td >66.74</td>
		</tr>
		<tr >
			<td ></td>
			<td >KCl</td>
			<td >0.5</td>
			<td >6.71</td>
		</tr>
		<tr >
			<td ></td>
			<td >CaCl2</td>
			<td >0.7</td>
			<td >6.31</td>
		</tr>
		<tr >
			<td ></td>
			<td >HEPES</td>
			<td >24</td>
			<td >100</td>
		</tr>
		<tr >
			<td ></td>
			<td >BSA</td>
			<td >15</td>
			<td >0.226</td>
		</tr>
		<tr >
			<td ></td>
			<td >Phenol Red</td>
			<td >0.01</td>
			<td >0.03</td>
		</tr>
		<tr >
			<td >Buffer 3, pH 7.4</td>
			<td >NaCl</td>
			<td >8</td>
			<td >137</td>
		</tr>
		<tr >
			<td ></td>
			<td >KCl</td>
			<td >0.35</td>
			<td >4.7</td>
		</tr>
		<tr >
			<td ></td>
			<td >MgSO4</td>
			<td >0.08</td>
			<td >0.66</td>
		</tr>
		<tr >
			<td ></td>
			<td >CaCl2</td>
			<td >0.18</td>
			<td >1.62</td>
		</tr>
		<tr >
			<td ></td>
			<td >HEPES</td>
			<td >2.4</td>
			<td >10</td>
		</tr>
		<tr >
			<td ></td>
			<td >BSA</td>
			<td >15</td>
			<td >0.226</td>
		</tr>
		<tr >
			<td >PBS, pH 7.4</td>
			<td >NaCl</td>
			<td >8</td>
			<td >137</td>
		</tr>
		<tr >
			<td ></td>
			<td >KCl</td>
			<td >0.2</td>
			<td >2.7</td>
		</tr>
		<tr >
			<td ></td>
			<td >Na2HPO4-7H2O</td>
			<td >1.15</td>
			<td >4.3</td>
		</tr>
		<tr >
			<td ></td>
			<td >KH2PO4</td>
			<td >0.2</td>
			<td >1.4</td>
		</tr>
		<tr >
			<td >Other reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Name</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr >
			<td >Percoll (PVP solution)</td>
			<td >GE Healthcare</td>
			<td >288555</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenase Type IV</td>
			<td >Sigma Aldrich</td>
			<td >C5138</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane&#160;</td>
			<td >Abcam</td>
			<td>ab144581</td>
			<td></td>
		</tr>
		<tr >
			<td >Hepatocyte Growth Medium</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Gibco</td>
			<td >11965118</td>
			<td></td>
		</tr>
		<tr >
			<td >10% by volume fetal bovine serum</td>
			<td >Gibco</td>
			<td >10437010</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen/Strep (2x)</td>
			<td >Gibco</td>
			<td >15140148</td>
			<td></td>
		</tr>
		<tr >
			<td >Long-term Hepatocyte Growth Medium</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td ></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >10% by volume fetal bovine serum</td>
			<td >Gibco</td>
			<td >10437010</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen/Strep (2x)</td>
			<td >Gibco</td>
			<td >15140148</td>
			<td></td>
		</tr>
		<tr >
			<td >Glucagon&#160;</td>
			<td >Sigma</td>
			<td>G3157-2mg</td>
			<td >14 ng/mL</td>
		</tr>
		<tr >
			<td >Insulin</td>
			<td >Sigma</td>
			<td>I9278-5mL</td>
			<td >0.5 U/mL</td>
		</tr>
		<tr >
			<td >Hydrocortisone</td>
			<td >Sigma</td>
			<td>H0888-1g</td>
			<td >7.5 mg/mL</td>
		</tr>
		<tr >
			<td >Epidermal Growth Factor</td>
			<td >BD Biosciences</td>
			<td>354001-100ug</td>
			<td >20 ng/mL</td>
		</tr>
		<tr >
			<td >LSEC medium</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI</td>
			<td >Gibco</td>
			<td >11875119</td>
			<td></td>
		</tr>
		<tr >
			<td >10% by volume fetal bovine serum</td>
			<td >Gibco</td>
			<td >10437010</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen/Strep (2x)</td>
			<td >Gibco</td>
			<td >15140148</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion

本协议说明了一种获得高产量和生存能力的小鼠肝细胞和正弦内皮细胞 (秒) 适合培养或获得细胞裂解物。在这个协议中, 门静脉被用作插管的部位, 而不是腔静脉, 因为这限制了最终肝脏制剂中其他可能的细胞类型的污染。在整个过程中不需要特殊的仪器。水浴作为热的来源, 以保持所有的缓冲和解决方案的温度。采用标准蠕动泵来驱动流体, 离心过程中需要一个冷冻台式离心机。这种技术的唯一限制是在门静脉内放置导管, 这对 18-25 克大小范围内的一些小鼠具有挑战性。这种技术的优点是只有一条静脉用于灌注, 而静脉的通路是快速的, 这可以最大限度地减少肝脏缺血和再灌注, 从而降低肝细胞的活力。这项协议的另一个优点是, 由于离心过程中细胞密度的不同, 人们很容易将活体与死肝细胞的视力区分开来。此协议中的单元格可以用于任何下游应用的细胞培养, 也可用于任何生化评估。

本文介绍了肝脏灌注和肝细胞和秒的纯化/富集的演示、精制方法, 为最佳细胞纯化/浓缩提供了与本参考文献中编写的其他打印报告类似的详细提示。复查5。确定细胞纯化成功的关键步骤出现在胶原酶灌注过程的路线和技术细节中, 并在本议定书中概述。与其他已发布11 (图 1) 的系统相比, 该设备的设置相当简单, 而且成本效益与标准的实验室仪器相对应。在这个设置中, 抱着鼠标的托盘坐在水浴中, 水浴中的一些多余的油管, 以保持在肝脏内的液体灌注的温度。这里所示的仪器可以用于小鼠和大鼠, 对大鼠肝脏灌注有稍微不同的几何12 (图 2)。
在这个过程中, 肝脏通过门静脉灌注而不是腔静脉 (这是另一种流行的灌注途径), 因为它在腹部内容易进入, 并且静脉直接流入肝脏。观察者应该知道, 门静脉有几个小分支, 可能短路的灌流液, 和导管应该被放置在这些分支的最佳成功13(图 3)。一旦门静脉被确定, 弯曲钳被用来绘制手术缝合或涤纶线下的门静脉在肝和上胰十二指肠静脉。该钳也用于理顺门静脉的积极血压 (图 4A)。导管内的针应平行放置在静脉旁 (图 4B), 斜面应轻轻插入静脉腔内 (图 4C)。正确的放置将显示沿导管出现的血液。一旦针被收回, 导管应稳定的线程绑在它和血压将迫使血液通过导管。建议将导管连接至泵浦油管, 然后血液泄漏 (图 4D)。一旦泵管连接, 立即切断一个主要的血管, 如腔静脉或主动脉腹型紫癜为血液/流体引流(图 5 a).通常需要切开老鼠腹壁的一侧, 以充分引流, 因为腹部的血液积聚使得难以想象灌注过程, 血液可以进行细胞纯化 (图 5 B). 一旦 PBS 开始灌注肝脏, 肝脏就会因缺血而漂白 (图 5C)。如果只有少数的裂片漂白, 那么可能的原因是导管被放置在肝脏太远, 需要慢慢地后退。一旦放置导管被确认, 使用防水胶带, 以确保油管到位。一般实验室胶带通常是不够的。为了确认导管的正确放置, 挤压血管以停止引流, 观察肝脏内的增加压力。这样做将有助于从肝脏冲洗血液 (图 5D)。当胶原酶溶液最初进入肝脏以确保所有裂片接触胶原酶时, 也应做到这一点。胶原酶溶液耗尽后, 良好的胶原酶消化是 Glisson 胶囊与软组织分离的指示。
细胞纯化的一般程序概述在图 6和图 7中, 其中肝细胞在低速旋转过程中早期就被收获, 在含 BSA 的缓冲区中4洗涤后非常纯净 (图8).秒与 KCs 在 PVP 梯度上 (图 9a) 进行了联合纯化, 然后通过选择性粘附在胶原涂层聚苯乙烯培养皿上进行分离。使用这种方法的秒纯度是 83-90% 纯, 主要取决于胶原酶消化的整体效果 (图 9B)。使用光显微镜进行定量测量 (肝细胞和秒都有区别于其他细胞的特征) 表明, 在一个代表性的制剂中, 肝细胞的纯度几乎是 100%, 而秒则仅为89% 纯 (表 1)。另外, 在 PVP 梯度后, 磁柱分离可以获得更高的秒富集, 尽管这超出了本协议的范围。关于秒和 KCs 的磁性分离的更多信息可能会在迈耶et . 中找到。9和刘et 。14应该记住的是, 肝细胞的生存能力在未充分消化的肝脏中迅速减少, 但对于 npc 来说, 这并不必要. 未充分消化的肝脏也产生更多的细胞碎片, 这不是完全在离心步骤中被淘汰。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56993/56993fig1.jpg"> 图 1: 灌注套件的示意图表示形式.烧瓶由夹子固定 (未显示);在过程中, 含有流体的油管从1L 烧瓶换成125毫升烧瓶, 由红色虚线表示。注意, 氧气饲料不会直接泡进烧瓶中的溶液中。瓶塞也应有缺口, 以允许封闭回路流体流动与油管插入在缺口。这在管材的热身期间和为所有空气的弹射在管材之内是关键的。空气流出是由连接到 Tygon 油管和夹紧钳的 T 连接器组成, 用于快速开启和关闭系统。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56993/56993fig2.jpg"> 图 2: 在小鼠肝脏灌注过程中的灌注套件.托盘与鼠标放在水浴的角落。氧与胶原酶溶液分离, 因为在热身过程中已经有氧。物品的位置是 A) 氧气线, B) 1 L 烧瓶含 PBS, C) 125 毫升瓶含有缓冲2与胶原酶, D) 泵, E) 泵控制, F) 气泡陷阱, G) 流体线运行从烧瓶, 通过泵和鼠标。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56993/56993fig3.jpg"> 图 3: 鼠标腹部血管的图像.插入导管应位于门静脉 (绿点) 的胃和胰静脉交界处以下, 尖端应放置在左、右肝门静脉 (黄点) 附近, 形成叉入肝脏的主裂片。一旦正确放置, 该导管应稳定与线程使用一个简单的上手结。K = 肾脏, L = 肝脏, SI = 小肠。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56993/56993fig4.jpg"> 图 4: 导管放置在门静脉中.(A) 钳用于确保门静脉的血液充血, 并使静脉导管的放置伸直。(B) 导管与门静脉平行排在一起, 并带有斜角。(C) 导管内针的斜角插入静脉, 而不是通过静脉。(D) 导管的针被缩回, 血液将通过导管的末端回流, 如镊子尖端所示。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56993/56993fig5.jpg"> 图 5: 正确的肝脏灌注.(A) 导管放置后, 导管的雌性鲁尔端连接到泵油管的雄性鲁尔端 (从1毫升注射器切割)。(B) 在切割一条主要降血管后, 血液和 PBS 从腹部排出, 用剪刀切开鼠标一侧。(C) 当血液被冲洗出来时, 肝脏应该会漂白, 并且 (D) 在压力下通过用镊子挤压切断血管而膨胀。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56993/56993fig6.jpg"> 图 6: 肝细胞纯化和鼻咽癌分离的示意图.大多数离心步骤的目的是从非实质细胞中移除活细胞和死肝脏。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56993/56993fig7.jpg"> 图 7: SEC 浓缩和纯化的示意图.npc 由 PVP 梯度分离, 其次是 SEC 分离, 短粘附在一个标准的聚苯乙烯板上。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56993/56993fig8.jpg"> 图 8: 纯化肝细胞.肝细胞被镀在胶原涂层的组织培养板上, DMEM + 8% 血清 + 笔/链球菌, 并孵化为6小时, 其次是图像采集显微镜下400X。酒吧等于20µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56993/56993fig9.jpg"> 图 9: SEC 纯化.(A) 从 25/50% PVP 接口 (黄色箭头) 中收集到秒和 KCs, 并使用无血清介质进行清洗。(B) 在 RPMI + 5% 的 KCs 中, 通过对标准培养皿进行选择性粘附、水洗和镀膜, 将该秒与该细胞分离。图像是在 400X EVOS 倒置显微镜拍摄的。酒吧等于20µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56993/56993fig10.jpg"> 图 10: 扫描电镜下小鼠肝脏解剖.小鼠肝脏用 PBS 灌流 (100 毫米常使用钠, 12 毫升25% 戊二醛, 15.6 毫升16% 多聚甲醛, 2.65 毫米氯化钙, 180.2 毫米蔗糖, 混合在100毫升溶液中), 以1毫升/分钟的速率进行切片, 并为扫描电子显微镜准备。在10,000X 的现场发射 SEM 上采集了图像。黄色箭头表示肝细胞之间的微绒毛。酒吧等于5µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56993/56993fig11.jpg"> 图 11: 可视化死与活肝细胞.2洗涤后与缓冲 1, 颗粒肝细胞可评估活/死比的眼睛。一个打火机颗粒 (左) 表明至少一半的肝细胞死亡。右侧较暗的颗粒更牢固地颗粒到管底, 超过90% 活。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56993/56993fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="3">表 1: 细胞纯化</td>
		</tr>
<tr>
<td>单元格类型</td>
			<td>% 目标单元格</td>
			<td>% 其他单元格</td>
		</tr>
<tr>
<td>肝</td>
			<td>99</td>
			<td>1</td>
		</tr>
<tr>
<td>Sinsusoidal 内皮细胞</td>
			<td>89。1</td>
			<td>10。9</td>
		</tr>
</tbody></table>表 1: 光镜对细胞纯度的评估。

Watch this Scientific Journal Video about Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion at JoVE.com

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

该协议的目的是获得高生存能力和高产量的肝细胞和正弦内皮细胞。这是通过灌注肝与 IV 型胶原酶溶液通过门静脉, 其次是差异离心获得肝细胞和正弦内皮细胞。

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

purification-hepatocytes-sinusoidal-endothelial-cells-from-mouse

Nanyang Technological University

Research

JoVE Journal

Biochemistry

50.0K 次观看.  University of Nebraska. 该协议的目的是获得高生存能力和高产量的肝细胞和正弦内皮细胞。这是通过灌注肝与 IV 型胶原酶溶液通过门静脉, 其次是差异离心获得肝细胞和正弦内皮细胞。

视频: Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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 ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.