Name: kupffer cell isolation for nanoparticle toxicity testing
Uploaded: 2015-08-18T14:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Kupffer Cell Isolation for Nanoparticle Toxicity Testing at JoVE.com

This work was supported by EU FP7-ITN Marie-Curie Network program RADDEL. MB and KAJ would like to acknowledge Joe Varguese and Rui Serra Maia for their help and suggestions along the optimization of the Kupffer cell isolation.

所有的动物实验均符合所有相关的准则，法规和监管机构执行。所演示的协议英国内政部监管的指导和批准下进行 1.灌注和细胞采集（图1） <img alt="图1" src="/files/ftp_upload/52989/52989fig1.jpg" /> 图1:肝灌注的小鼠的麻醉之后，消化道横向移动到腹部的左侧，以使门静脉（PV）的访问。光伏使用的EGTA / HBSS溶液缓慢的流速（1-3毫升/分钟）和下veina静脉（IVC）是空心立即破裂，以避免肝脏内的任何超压。内灌注的第一分钟，流速逐渐增加至7毫升/分。胶原酶溶液，然后灌注以10毫升/分钟，直到其完全消化的实现。 <ol><li>编写，J再新鲜的材料表中描述的所有试剂。 </li><li>暖EGTA（乙二醇四乙酸）/ HBSS（Hank氏平衡盐溶液）溶液（每只小鼠50ml）和胶原酶溶液（每只小鼠100毫升）中30分钟，在40℃。 </li><li>首先用70％乙醇冲洗泵软管。倾40毫升EGTA / HBSS溶液到离心管浸在水浴中，并冲洗泵软管与预热EGTA / HBSS解。 </li><li>使用巴比妥类药物的呼吸抑制而死亡之前，可靠地生产无意识进行终端麻醉。苯巴比妥注射1毫克/千克，IP为女性或男性CD1小鼠（35-45克）。确认由麻醉捏脚趾。 </li><li>剃腹部毛和消毒用70％的乙醇溶液中的腹部表面。 </li><li>切断通过腹腔和通过横向移动肠道到腹部的左侧露出门静脉和下腔静脉。 </li><li>明星T中的泵以1-3毫升/分钟，EGTA / HBSS解决方案的速度和导管插入使用23克蝴蝶针门静脉（翅切）。夹住门静脉插管与23政针的部分，然后翻转serrefine镊子在打开小鼠腹部的表面上。肝脏应该在前30秒EGTA / HBSS解决方案灌注内迅速苍白。 </li><li>快速切割下腔静脉的下部，以避免在肝脏过量压力建筑物，再逐渐增加的流速至7毫升/分钟，在灌注的第一分钟。动物死亡是由于出血继发腔静脉穿刺。 </li><li>当降到低于5毫升EGTA / HBSS解决方案是将剩余的离心管，填补它与胶原酶溶液（40-50毫升）。逐渐增加的流速10毫升/分钟，在30秒内。 </li><li>通过将压力施加到下腔静脉 ，使用镊子，5-10秒的间隔使肝脏膨胀。 THIS能周期性地进行（在消化过程中的5-10倍）。此步骤会改善肝细胞​​解离，降低了灌注时间用胶原酶。 </li><li>当小于10毫升胶原酶溶液保留在离心管内，倒入40-50预热的胶原酶溶液放入离心管中。 </li><li>后灌注10-15分钟，约70-80毫升胶原酶溶液灌注，应用用钳子肝脏的表面上的小的压力。压力的打印指示肝细胞解离。 </li><li>从腹腔取出肝作为一件，并将其放置在含有20-30毫升库普弗细胞分离培养基的离心管中。保持肝细胞在冰上或4℃下进行3小时的最大时间段，以避免影响肝细胞活力。 </li><li>上重复，如果需要几个动物的灌注过程中，同时保持灌注肝脏在冰上。游泳池差一分三时肝脏净化并继续第3阶段。 </li></ol> 2.制备的密度梯度离心的（图2） <img alt="图2" src="/files/ftp_upload/52989/52989fig2.jpg" /> 图2:密度梯度的制备所有步骤均在无菌条件下进行。所述SIP通过混合15.3毫升涂布二氧化硅粒子的溶液1.7毫升10×PBS中制备。将5ml的SIP都用15毫升的PBS混合以赚20毫升25％的SIP溶液。 10毫升SIP都用10毫升的PBS混合以赚20毫升50％的SIP溶液。缓慢加入用血清学25毫升吸管含有50％的SIP溶液（20毫升）的倾斜，并使25％的SIP溶液（20毫升）的离心管中。 <ol><li>继续在无菌条件下以下步骤（部分2，3，5，和），并保持细胞在冰上或4℃。通过混合15.3毫升涂布二氧化硅粒子溶液1.7毫升10×PBS准备SIP（等渗包覆二氧化硅粒子溶液）。 </l我&gt; <li>为了赚20毫升25％的SIP解决方案，混合5毫升SIP用15毫升的PBS。为了赚20毫升50％的SIP解决方案，混合10毫升SIP用10毫升的PBS。 </li><li>填空的话就是离心管20毫升的50％的SIP解决方案。倾斜离心管以一个角度接近90°，在侧壁的管慢慢加入25％的SIP溶液（20毫升）用25毫升血清吸管，而不触及50％的SIP层的表面上。当加入25％的SIP溶液，逐步减少管的角度。保持密度梯度在冰上直到使用。 </li></ol> 3.库普弗细胞的纯化（图3） <img alt="图3" src="/files/ftp_upload/52989/52989fig3.jpg" /> 图3:库普弗细胞通过密度梯度离心和细胞粘附的纯化所有步骤均在无菌条件下进行。 （A）中的Glisson囊破裂后，肝细胞通过一个1过滤00微米的过滤器。将悬浮液离心×50克至丢弃肝细胞（粒料），并收集非实质细胞部分（上清液）。这个步骤重复3次。非实质细胞加入上不连续等渗梯度的顶部，并在800×g离心离心15分钟。从25％的SIP坐垫收集库普弗细胞通过细胞粘附的选择进一步纯化。 （B）的细胞接种于24孔板中并在37℃和5％CO 2的30分钟。非贴壁细胞用500微升的HBSS洗涤一次。 Kupffer细胞显示镀层，当f -CNTs可以加入到细胞中后，其贴壁形态学4小时。比例尺为25μm。 <a href="https://www.jove.com/files/ftp_upload/52989/52989fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>放一灌注肝脏在培养皿用15ml枯否细胞分离培养基。破裂格利森囊（用剪刀和释放所有肝细胞进入枯否细胞分离介质肝脏膜）。过滤溶液，虽然以被收集在一个离心管中100微米的细胞滤网。重复上附加灌注肝脏的灌注过程和（从三只小鼠从一个鼠标15毫升，高达45毫升）中汇集的细胞以相同的离心管中。 </li><li>离心细胞悬浮液，在50×g离心在4℃下2分钟。实质细胞（肝细胞）将在颗粒和非实质细胞（包括Kupffer细胞）将在上清液中。 </li><li>收集在一个干净的离心管中，离心上清，在50×g离心每2分钟。重复此步骤三次。 </li><li>离心机在1350×g离心15分钟以沉淀非实质细胞。弃上清，悬浮颗粒在10毫升枯否细胞分离培养基。 </li><li>添加的非实质细胞溶液的不连续等渗梯度五十分之二十五％，如在2.3中描述。一分钱rifuge在850 XG 15分钟没有加速或休息。 </li><li>本地化的25％的SIP分数接近25/50％SIP接口（ 图3）内出现混浊的富集库普弗细胞级分。使用10ml的血清吸液管，吸出约12毫升富集库普弗细胞级分的。转移细胞到含35-40毫升库普弗细胞分离培养基的离心管中。轻轻混匀离心机在1350×g离心15分钟，在4℃以沉淀细胞。 </li><li>丢弃在5-10毫升预热的枯否细胞培养基的上清液和重悬细胞。计数细胞（血球），并使用台盼蓝染色法测定存活率。 </li><li>板的纯化的非实质细胞在24孔板在5×10 5细胞/孔的密度。孵育细胞在37℃和5％的CO 2（ 图3）。 30分钟后，轻轻取出介质，洗涤预热Hank氏平衡盐溶液（HBSS）一次，然后更换它用500μl新鲜的和预温热库普弗细胞培养基（每孔）。 </li><li>离开Kupffer细胞至少4小时，在37℃和5％的CO 2治疗之前，纳米颗粒，以使它们能够获得他们附着的形态。 </li></ol> 4.表征<ol><li>枯否细胞纯度<ol><li>准备用于保持库普弗细胞活力新鲜的PBS / BSA溶液。用500μl的PBS / BSA溶液洗一次细胞。取出PBS / BSA溶液分离枯否细胞在500微升新鲜的PBS / BSA溶液用温柔刮擦。当细胞置于24孔板中，缩短刮刀的末端用剪刀，使脱离更容易进行。传递细胞成流式细胞仪管。 </li><li>计数使用血球和离心机1×10 5个细胞在1350×g下Kupffer细胞。重悬Kupffer细胞于30μlF4 / 80抗体（纯）的。拌匀，并在室温下孵育30分钟。保持在冰上的未染色细胞（未孵育的抗体）。 </li><li>加入2毫升的PBS / BSA溶液中染色的细胞，离心在1350 g下5分钟，弃去所得的上清液。悬浮在200μlPBS / BSA的染色细胞。 </li><li>用于流式细胞术分析，栅万根据它们的大小（前向散射）和粒度（侧向散射）不染色Kupffer细胞。在FL-1通道分析门控细胞的荧光。 </li><li>用同样的方法染色枯否氏细胞保持用于未染色术枯否细胞分析流程相同的设置。选择和量化染色细胞的荧光来评估枯否细胞的纯度。 </li></ol></li><li>枯否细胞吞噬活性<ol><li>用0.5％更换培养基（体积/体积）中的枯否细胞培养基的荧光珠孵育4小时，在37℃和5％的CO 2。 </li><li>为了在1350×g离心，二除去非内化珠，离心机Kupffer细胞SCARD上清，悬浮细胞在500μlPBS / BSA溶液。重复此步骤2次以上。 </li><li>刮枯否细胞在500微升PBS / BSA解决方案和转移的细胞成流式细胞仪管。 </li><li>离心机枯否细胞在1350 XG，丢弃在200微升PBS / BSA溶液的上清，重悬细胞。 </li><li>用于流式细胞术分析，栅万根据它们的大小（前向散射）和粒度（侧向散射）不染色Kupffer细胞。在FL-2通道门控分析细胞的荧光。 </li></ol></li></ol> 5.孵化化学官能化的碳纳米管（六-CNTs） <ol><li>通过超声处理在使用前15分钟，制备˚F-CNTs（1毫克/毫升）的水分散体:F -CNTs制备在内部22。 </li><li>准备2倍浓缩碳纳米管F- 分散枯否细胞培养基。 分散在介质中超声处理F-碳纳米管在进行步骤5.3前2分钟）。 </li><li>对于每个孔，用250微升2倍˚F-CNTs分散取代250微升旧媒体。未处理孔（250微升条件培养基+ 250微升新鲜库普弗细胞培养基的）中，用10％DMSO处理的孔用作阴性和阳性对照，分别为。 </li><li>孵育Kupffer细胞在37℃和5％CO 2的24或72小时。 </li></ol>在枯否细胞˚F-CNTs通过修改乳酸脱氢酶（LDH）测定6毒性评估<ol><li>弃去上清液。 </li><li>加入裂解缓冲液的200微升（每24孔板孔）并在37℃下进行30分钟-1小时。 </li><li>进行剧烈吸移后，收集裂解Kupffer细胞成离心管和离心机以40,000×g离心10分钟沉淀物溶解由细胞和细胞碎片使得f -CNTs。 </li><li>收集上清（ƒ-CNT免费）到离心管并在-20℃储存或继续执行步骤6.5）。 </li><li>转移50微升到96孔板中并加入底物混合物溶液（LDH试剂盒）等体积的各孔中。覆盖该平板，加入50μl的终止溶液（LDH试剂盒）之前在室温下孵育15分钟。添加含有50微升裂解缓冲液，50微升底物混合物溶液和50微升的终止溶液空白孔的一式三份。 </li><li>读取490nm处的吸光度在酶标仪并使用以下公式来计算（％）细胞活力。 </li></ol><img alt="式（1）" src="/files/ftp_upload/52989/52989eq1.JPG" />

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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+Intact+Liver+Cells.">Isolation of Intact Liver Cells.</a> Nature. 180 (4597), 1283-1284 (1038).</li><li>Smedsr&#248;d, B., Pertoft, H. <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+pure+hepatocytes+and+reticuloendothelial+cells+in+high+yield+from+a+single+rat+liver+by+means+of+Percoll+centrifugation+and+selective+adherence.">Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.</a> Journal of Leukocyte Biology. 38 (2), 213-230 (1985).</li><li>Morin, O., Patry, P., Lafleur, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+endothelial+cells+of+adult+rat+liver+as+resolved+by+sedimentation+velocity+and+flow+cytometry.">Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry.</a> J Cell Physiol. 119 (3), 327-334 (1984).</li><li>Miller, S. 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=Purification+of+cells+from+livers+of+carcinogen-treated+rats+by+free-flow+electrophoresis.">Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.</a> Cancer Res. 43 (9), 4176-4179 (1983).</li><li>Firme, C. P., Bandaru, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+issues+in+the+application+of+carbon+nanotubes+to+biological+systems.">Toxicity issues in the application of carbon nanotubes to biological systems.</a> Nanomedicine. 6 (2), 245-256 (2010).</li><li>Ali-Boucetta, H., Al-Jamal, K. T., Kostarelos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+assessment+of+carbon+nanotube+interaction+with+cell+cultures.">Cytotoxic assessment of carbon nanotube interaction with cell cultures.</a> Methods in Molecular Biology. 726, 299-312 (2011).</li><li>Wang, 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=Understanding+and+correcting+for+carbon+nanotube+interferences+with+a+commercial+LDH+cytotoxicity+assay.">Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.</a> Toxicology. 299 (2-3), 99-111 (2012).</li><li>Wang, J. T. -. 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=Magnetically+Decorated+Multiwalled+Carbon+Nanotubes+as+Dual+MRI+and+SPECT+Contrast+Agents.">Magnetically Decorated Multiwalled Carbon Nanotubes as Dual MRI and SPECT Contrast Agents.</a> Advanced Functional Materials. 24 (13), 1880-1894 (2014).</li><li>Li, P. Z., Li, J. Z., Li, M., Gong, J. P., He, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+to+isolate+and+culture+mouse+Kupffer+cells.">An efficient method to isolate and culture mouse Kupffer cells.</a> Immunol Lett. 158 (1-2), 52-56 (2014).</li><li>Froh, M., Konno, A., Thurman, R. G. <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+liver+Kupffer+cells.">Isolation of liver Kupffer cells.</a> Curr Protoc Toxicol. Chapter 14, Unit14 14 (2003).</li></ol>

The authors declare that they have no competing financial interests.

下面的步骤是关键的，以实现高产量和枯否氏细胞的高存活率。在无菌条件应用于限制细菌和真菌污染的风险。所有仪器使用前需要灭菌。试剂应在进行分离步骤之前新鲜制备。 Ⅳ型胶原酶的选择，具有低胰蛋白酶的活性，是至关重要的。不同批次来自同一供应商具有不同的酶活性和它们可能需要最初比较，以便选择最合适的批次库普弗细胞分离。这可以通过评估细胞产量?...

纳米毒理学研究领域的目的是表征纳米颗粒的生物效应。基于在体内研究毒理学研究仍然是最精确的方法。然而，它们的使用受到它们的成本，劳动和时间要求1限定。作为替代， 在体外测定法已被使用，因为它们的简单性和开发高通量体外测试平台2的可能性-如此延伸的测试条件的数量。最nanotoxicological研究使用体外测定与永生化的细胞系进行。然而，也有关于这些实验的结果外推到体内毒理学影响3担忧。事实上，永生化细胞系的性质可以是显著不同从组织它们衍生自， 例如 ，遗传转化4，关键形态特征恶化5，细胞极性6和功能的改变，如炎症介质7的调节的丧失。 Kupffer细胞是最丰富的巨噬细胞群中的主体，并直接与血液接触由衬肝血窦的壁上。作为网状内皮系统（RES）的一部分，这些巨噬细胞是负责循环纳米颗粒和因此的捕获，构成一个非常合适的模型来研究纳米颗粒的毒性。 体内 8 并在体外与暴露于纳米粒子枯否氏细胞相关的炎症反应9研究已在其他地方发表。枯否细胞也参与肝脏疾病的发病机制，如酒精性肝病10，肝纤维化11或病毒性肝炎12。据报道，孤立的枯否细胞提供了有益的启示来形容细胞机制INVolved肝破裂13,14。 几种方法已经报道了分离和纯化Kupffer细胞。细胞的分离可导致机械或酶解15。胶原酶消化节约表明枯否细胞的功能完整性，而导致高枯否细胞产量16的优势。许多实验方法，在复杂性和成本不同，已用于Kupffer细胞从其他肝细胞群中分离出来。例如，枯否细胞纯度可通过免疫亲和17，流电泳18，选择性粘附16或通过离心技术16，根据它们的大小和密度而选择的细胞来实现。这些方法的组合可以被选择为增加的人口16的纯度。没有关于对枯否细胞分离的理想方法共识的，因为它主要取决于应用和可用的equipmen吨。然而，在纳米颗粒的毒性测试的情况下，简单性和高收率与Kupffer细胞的功能保存相关的技术似乎是对本申请中最适合的。 这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法是从由Smedsrød 等人开发的原始协议进行修改。16设计用于大鼠肝细胞的分离。大多数研究报告，并从大鼠肝脏描述Kupffer细胞的分离。在此，我们描述了一种方法，从小鼠肝脏分离Kupffer细胞，以高收率和纯度。使用小鼠降低了实验成本，并允许几个肝脏的处理，以获得纳米颗粒的毒性测试大量Kupffer细胞。 在下面的协议，枯否细胞与官能化的碳纳米管（六</EM&gt; -CNTs）。碳纳米管的独特的物理化学性质- 即高长径比和表面积大，都取得了碳纳米管有趣候选作为用于治疗和诊断目的的载体。然而，忧虑已提出了关于碳纳米管19的毒性，以及新的体外测试的发展旨在提高CNT生物效应的理解。在枯否细胞毒性与缺乏细胞膜的结构完整性有关。这是通过胞质酶LDH的损失从细胞到上清液计量。这种方法的原理，因此，是除去任何释放LDH和测量什么留在电池20。这在要求完成测量所释放的LDH在上清液因为CNT的上清液中的存在干扰测定21。 我们建议使用这种简单和成本效益枯否细胞的分离实现方法具的d，来分离大量的功能枯否细胞。这允许范围的纳米颗粒的毒性的筛选，在一个相关的初级巨噬细胞的模型。

<table><tbody><tr><td>Euthatal (pentobarbital sodium)</td><td>Merial</td><td></td><td></td></tr><tr><td>CD1 mice</td><td>Charles River&amp;nbsp;&amp;nbsp;</td><td>-</td><td>Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (&lt;em&gt;e.g.&lt;/em&gt; a male CD1 mouse of 7 weeks old exceeds 35 g in weight). It is also advised to contact the animal supplier in advance to arrange for the delivery of older animals. Alternatively animals can be in-house to reach the desired weight.&amp;nbsp;</td></tr><tr><td>HBSS (Ca&lt;sup&gt;2+ &lt;/sup&gt;and Mg&lt;sup&gt;2+ &lt;/sup&gt;free, with bicarbonate)&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>14175-053</td><td>HBSS must be&amp;nbsp; Ca&lt;sup&gt;2+&lt;/sup&gt; free.&amp;nbsp;</td></tr><tr><td>Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>E8145&amp;nbsp;&amp;nbsp;</td><td>EDTA can also be used but EGTA has the advantage of chelating Ca&lt;sup&gt;2+ &lt;/sup&gt;selectively.&amp;nbsp;</td></tr><tr><td>HEPES (1 M)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>15630-056&amp;nbsp;</td><td>100 ml&amp;nbsp;</td></tr><tr><td>Collagenase type IV&amp;nbsp;</td><td>Worthington&amp;nbsp;</td><td>CSL-4&amp;nbsp;</td><td>It is advised to test different batches of collagenase or at least mention to the supplier the product has to be suitable for liver cell isolation&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Low glucose DMEM&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>D5523&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>RPMI (with sodium pyruvate and Glutamax)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>12633-012&amp;nbsp;</td><td>500 ml</td></tr><tr><td>Penicillin/Steptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>15140-122</td><td>100 ml</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>First-Link&amp;nbsp;&amp;nbsp;</td><td>60-00-850&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>Trypan blue solution&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8154&amp;nbsp;</td><td>100 ml&amp;nbsp;</td></tr><tr><td>Coated silica particle solution (Percoll)</td><td>GE Healtcare&amp;nbsp;&amp;nbsp;</td><td>17-0891-02&amp;nbsp;</td><td>Percoll&amp;reg; is very stable and can be kept for several years&amp;nbsp;</td></tr><tr><td>1x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>10010-023</td><td>500 ml&amp;nbsp;</td></tr><tr><td>10x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>70011-036</td><td>500 ml</td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>D/4120/PB08&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>LDH kit (CytoTox 96)&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>G1781&amp;nbsp;</td><td>Keep protected from light&amp;nbsp;</td></tr><tr><td>DMEM phenol red free</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>31053-028&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>F4/80 antibody (Alexa 488)</td><td>AbD Serotec</td><td>MCA497A488</td><td>Do not dilute, used neat for flow cytometry</td></tr><tr><td>Fluorescent beads</td><td>Sigma</td><td>L2778</td><td>Latex beads, amine-modified polystyrene, fluorescent red. 1 ml</td></tr><tr><td>Name of the Material</td><td>Company&amp;nbsp;</td><td>Catalog number&amp;nbsp;</td><td>Comments/Description&amp;nbsp;</td></tr><tr><td>Butterfly blood collection set (23 G/305 mm long tubing)&amp;nbsp;&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>367288</td><td></td></tr><tr><td>Syringe Filters (0.22 &amp;mu;m Blue Rim)&amp;nbsp;</td><td>Minisart&amp;nbsp;</td><td>16534-K&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge Tubes (50 ml Blue Cap)&amp;nbsp;</td><td>BD Biosciences, Falcon&amp;nbsp;</td><td>35 2070&amp;nbsp;</td><td></td></tr><tr><td>Petri dish (90 x 15 mm)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>BSN 101VR20&amp;nbsp;</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer&amp;nbsp;&amp;nbsp;</td><td>BD&amp;nbsp;&amp;nbsp;</td><td>352360</td><td></td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>Watson Marlow&amp;nbsp;</td><td>SciQ 300&amp;nbsp;&amp;nbsp;</td><td>Rinse tubing before and after each usage with sterile PBS and 70% ethanol.</td></tr><tr><td>24-well plates</td><td>Corning&amp;nbsp;&amp;nbsp;</td><td>3526</td><td></td></tr><tr><td>96-well plates&amp;nbsp;</td><td>Corning&amp;nbsp;&amp;nbsp;</td><td>3595</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Plate reader</td><td>BMG Labtech</td><td>FLUOstar Omega</td><td></td></tr><tr><td>Serrefine forceps</td><td>Hammacher GmbH&amp;nbsp;</td><td>Art. Nr. HSE 004-35 / Cat. Nr. 221-0051</td><td>The serrefine forceps allow to clamp the vessel cannulated with the 23 G needle without the need of holding the forceps during the perfusion procedure. URL: (http://www.hammacher.de/Laboratory-Products/Clamps-forceps/Serrefines/HSE-004-35-Serrefine::25126.html)&amp;nbsp;</td></tr><tr><td>Flow cytometry tubes</td><td>BD Biosciences, Falcon&amp;nbsp;</td><td>352052</td><td></td></tr><tr><td>Microcentrifuge tubes (1.5 ml)</td><td>Elkay</td><td>&amp;nbsp; 000-MICR-150</td><td></td></tr><tr><td>Cell scraper</td><td>BD Biosciences, Falcon</td><td>353086</td><td>Cut blade extremities with a pair of scissors to scrape cells in 24-well plates.</td></tr><tr><td>Name of the Reagent</td><td>Company&amp;nbsp;</td><td>Catalog number&amp;nbsp;</td><td>Comments/Description&amp;nbsp;</td></tr><tr><td>&amp;nbsp;EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank&#039;s Balanced Salt Solution) Solution&amp;nbsp;</td><td></td><td></td><td>HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Collagenase Solution&amp;nbsp;</td><td></td><td></td><td>DMEM low glucose containing collagenase type IV at 100 UI/ml, 15 mM HEPES and 1% Penicllin/Streptamycin (v/v). Adjust pH to 7.4. Prepare 100 ml for each liver to perfuse. After adding the collagenase, it is advised to warm up the solution for 30 min before use. This allows the collagenase activity to be optimum.&amp;nbsp;</td></tr><tr><td>Kupffer Cell Isolation Medium&amp;nbsp;</td><td></td><td></td><td>RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax&amp;reg; (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Kupffer Cell Culture Medium&amp;nbsp;</td><td></td><td></td><td>RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax&amp;reg; (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>SIP (solution of isotonic coated silica particles)</td><td></td><td></td><td>Mix 1.7 ml of 10x Phosphate Buffered Saline&amp;nbsp; with 15.3 ml of Percoll&amp;reg; to obtain 17mL of SIP.</td></tr><tr><td>25% SIP solution&amp;nbsp;</td><td></td><td></td><td>Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td></tr><tr><td>50% SIP solution&amp;nbsp;</td><td></td><td></td><td>Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline&amp;nbsp;</td></tr><tr><td>Lysis Buffer</td><td></td><td></td><td>DMEM media with 0.9% Triton X-100</td></tr><tr><td>PBS/BSA Solution</td><td></td><td></td><td>Prepare fresh Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin.&amp;nbsp;</td></tr></tbody></table>

kupffer cell isolation for nanoparticle toxicity testing

绝大多数体外 nanotoxicological研究已经使用永生化细胞系为他们的实用性。然而，从纳米颗粒的毒性测试结果在永生化细胞系或原代细胞已经显示不符，强调需要延长使用的原代细胞的体外测定法。这个协议描述了小鼠肝巨噬细胞的分离，命名Kupffer细胞，以及它们的使用来研究纳米颗粒的毒性。 Kupffer细胞是最丰富的巨噬细胞群在体内和构成网状内皮系统（RES），负责循环纳米颗粒的捕获部分。这里报道的枯否细胞的分离方法是基于一种两步灌注法进行纯化上密度梯度。该方法的基础上，胶原酶消化和密度离心，适于从由Smedsrød 等人开发的原始协议。专为大鼠肝细胞的隔离和n和提供高产率（每鼠14×10 6个细胞）和枯否细胞的高纯度（&gt; 95％）。这种隔离方法不需要复杂的或昂贵的设备，因此代表的复杂性和细胞产量之间的理想折衷。使用较重的小鼠（35-45克）提高了分离方法的产量，但也有利于显着的门静脉插管的过程。的官能化的碳纳米管˚F-CNTs毒性测定在该模型由改性的LDH测定。该方法通过测量缺乏的枯否细胞膜结构完整性孵育以f -CNTs后评估细胞生存力。毒性反应为f -CNTs可以使用一致此法来衡量，强调指出，孤立的枯否细胞对纳米颗粒的毒性测试非常有用。纳米毒理学的整体理解可以受益于这种模式，使得纳米颗粒的选择临床翻译更多艾菲cient。

非纯化的实质细胞的数目是一致的和远程每只小鼠之间8和14×10 6个细胞（17隔离被执行）。每只小鼠隔离足以板16〜28的孔中。通过台盼蓝染色细胞存活率显示出细胞存活率〜95％。库普弗细胞显示内温育30分钟的圆形状，在37℃，与它们的不完全粘合的形态（ 图4A）。在4小时温育，之后，将细胞分散和细胞簇开始形成。 然后枯否细胞的特点，以确认其纯度...

Watch this Scientific Journal Video about Kupffer Cell Isolation for Nanoparticle Toxicity Testing at JoVE.com

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

枯否细胞分离的纳米粒子毒性测试

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

kupffer-cell-isolation-for-nanoparticle-toxicity-testing

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Immunology and Infection

23.6K 次观看.  King's College London. Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells. 

视频: Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

去除微量元素的氧化铜纳米粉体制备铀<em&gt;原位</em&gt;恢复泌水及其对细胞活力的影响

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

科研

环境科学

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

免疫与感染

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工程

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

癌症研究

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

小胶质细胞作为替代生物传感器确定纳米颗粒神经毒性

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

神经科学

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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 ...

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 ...

生物学

Isolation and Culture of Mouse Kupffer Cells

Source: Bourgognon, M., et al. <a href="https://app.jove.com/v/52989">Kupffer Cell Isolation for Nanoparticle Toxicity Testing.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of Kupffer cells or liver macrophages from the mouse liver. The liver is perfused with a digestion solution followed by density gradient separation. The isolated adherent Kupffer cells can be used for further analysis.

小鼠 Kupffer 细胞的分离和培养

Source: Bourgognon, M., et al. Kupffer Cell Isolation for Nanoparticle Toxicity Testing. J. Vis. Exp. (2015)
In this video, we demonstrate the isolation ...