Name: isolation of cd146+ resident lung mesenchymal stromal cells from rat lungs
Uploaded: 2016-06-17T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs at JoVE.com

JJPC is supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship. MAM receives a merit scholarship from the German National Academic Foundation - Studienstiftung des Deutschen Volkes and is supported by a grant from the EFCNI (European Foundation for the Care of the Newborn Infant). BT is supported by CIHR, the Canadian Lung Association, the Stem Cell Network and the Children's Hospital of Eastern Ontario Research Institute.

所有的程序是由渥太华大学的动物护理委员会（动物伦理协议OHRI-1696）的批准。动物保健是按照机构的指导进行。 1.肺间质干细胞的分离<ol><li>在50毫升管准备酶混合物:权衡30单位中性蛋白酶2500ü我胶原酶和500üDNA酶一，这些款项足以满足成年小鼠或大鼠小狗的肺部。在4℃直至使用上隔离和存储的天制备。 </li><li>牺牲幼鼠第13天通过腹膜内注射戊巴比妥钠（0.2毫升，65毫克/毫升）。用脚趾捏反射建立神志不清。动物的死亡是通过打开胸部保证，概述如下。 </li><li>通过用70％乙醇喷雾动物消毒皮肤，小心使用手术剪，开始在膜片和朝喙侧切割打开胸廓，非常小心地不损伤肺部。传播胸腔打开使用止血钳，或者切掉胸腔以提供进入胸腔。 </li><li>通过移除心脏抽血动物。要取出心脏，把握胸腺和心脏小镊子和手术剪刀剪这些东西拿走。紧接着，吸收血液用纱布，直到没有更多的血液出来切断的主动脉和肺动脉的。 </li><li>从胸腔取出肺脏如下:持用小镊子气管，然后断绝与上喙侧手术剪气管。同时轻轻拉动肺包出来的胸部，切掉任何结缔组织沿肋骨背侧释放肺部。 </li><li>通过沿与手术剪膜片切割切断从主动脉和食管肺部。现在，肺是完全从胸部自由用纱布轻轻除去任何残留的血液。 </li><li>取出气管和支气管与河畔gical剪刀和仔细的肺叶转移到含冷35毫升30％柠檬酸盐 - 磷酸盐 - 葡萄糖腺嘌呤（CPDA-1）抗凝50ml管（26.30克柠檬酸三钠二水合物，3.27克抗坏血酸一水合物，2.22加入磷酸一钠，磷酸二氢钾， 31.80克D-葡萄糖，在1L0.275克腺嘌呤纯化的 H 2 O;无菌过滤的磷酸盐缓冲盐水（PBS），使用在使用前0.22μm的膜滤器）以除去血液和碎屑的溶液。 </li><li>在30％CPDA-1 / PBS中的5分钟的冲洗后，肺转移到含有35毫升无菌磷酸盐缓冲盐水（PBS）（RT）下一个新的50ml管中，倒置轻轻除去柠檬酸盐。转移到含有管35毫升Dulbecco氏PBS +钠丙酮酸+葡萄糖（DPBS ++）（RT）。每次清洗步骤大约需要5分钟。 注意:DPBS +可商购订购或由如下21:氯化钙二水合物132.4毫克，氯化镁六水合物100毫克，氯化钾（无水）200米克，磷酸二氢钾（无水）200毫克，氯化钠（无水）8000毫克，磷酸氢二钠（无水）1,144.5毫克，D-葡萄糖1000mg的，丙酮酸钠36毫克在1L纯净H 2 0，pH值7.3。无菌过滤器使用在使用前0.22μm滤膜过滤该溶液。 </li><li>通过加入10ml DPBS + +（预热至37℃）并轻轻翻转直至溶解溶解酶混合物。 </li><li>转移肺部到一个新的50ml管中，并用手术刀切碎在管的壁的肺直到切碎。酶组合加入至组织（10毫升酶混合物/每成年小鼠/大鼠小狗肺），盖紧管和在38℃，轻轻摇动孵育（无论是在热混合器，振荡水浴，或水与常规的手动搅拌槽）。持续时间取决于组织类型:胎儿组织60分钟，幼年/成人组织90-120分钟，纤维化成人组织120-150分钟。 </li><li>通过螯合二价阳离子W停止消化第i 200微升乙二胺四乙酸（EDTA，0.15M，pH为7.4。无菌过滤器在使用前用0.22微米的膜过滤器的溶液）。 </li><li>加入20毫升5％胎牛血清（FBS）/ PBS中，并通过100微米的细胞滤网整个悬浮液传递到一个新的50ml管中。冲洗管和细胞过滤用10毫升5％FBS / PBS中，使总体积至40ml。 </li><li>离心在500 xg离心，室温下5分钟。 </li><li>除去上清液（沉淀应清晰可见），悬浮在40毫升5％FBS / PBS，重复离心步骤1.13。 </li><li>悬浮在4ml 5％的FBS / PBS（RT）和层小心以上3毫升密度梯度介质（1.073克/厘米3）在一个15毫升管14。以获得良好的相间，至关重要的是，在单细胞悬浮液的分层发生在以非常低的速度&gt; 45°角。 </li><li>在900 XG离心20分钟，中（5/10）加速，减速没有，19℃。 </li><li>收集存在于细胞中的terphase和重悬在10ml无菌PBS。 </li><li>离心机在500 XG 5分钟，21℃。 </li><li>去除上清，重悬细胞沉淀在10毫升预热完成了L-MSC培养基（最低基本介质鹰，阿尔法修改（αMEM），辅以每500毫升αMEM，20％体积/体积FBS 2毫摩尔L-谷氨酰胺和1％体积/体积青霉素/链霉素/两性霉素B. </li><li>重复离心步骤1.18。随后，去除上清，重悬在10毫升预热的新鲜培养基。 </li><li>算使用血球或自动细胞计数器和板以高密度的细胞，因为在此阶段的大多数细胞都没有塑料贴壁。确切的铺板密度取决于动物种类和年龄，例如成年小鼠肺的细胞以10 5个细胞/ cm 2接种，而从大鼠小狗肺细胞是足够致密以1-2接种密度点¯x 10 4个细胞/ cm 2的</sup&gt;。使用0.2毫升L-MSC培养基/ cm 2以下。 </li><li>培养L-的MSC在湿润的5％ 的 O 2，5％的CO 2气氛中于37℃。 </li><li>用PBS洗涤一次后更改初始播种后媒体24小时，每3天以后。细胞应该是80-90，直到％汇合，这需要3-5天，平均取决于原产地的动物养殖。较高的汇合将促进分化为成纤维细胞。 </li></ol> 2. CD146 +肺间质基质细胞的选择<ol><li>二抗磁珠涂料<ol><li>涂层在圆底磁珠用生物素化的次级抗体，在10微克的二级抗体/ 100微升磁珠的比率无菌的毫升无菌条件离心管。使用10微升的二级抗体每预期的0.5×10 6个细胞 ，并用最小100微升磁珠和10μg抗体。 注:一个比每珠使用量可能制造商之间差异tibodies，请查阅制造商的说明选择的珠子。对于这个协议，请参考表1对该协议的优化中使用的珠子和抗体。 </li><li>孵育在一个旋转样品混合器在30转/分钟（RPM），在室温30分钟。 </li><li>重悬珠子在1.5ml的无菌0.1％牛血清白蛋白（BSA）/ PBS中，并转移到3毫升圆底管（流式细胞术管）。冲洗冷冻管与另外1.5毫升0.1N％BSA / PBS和转移到3毫升管。 </li><li>放置在适当的磁体的管，并等待2分钟，直到所有的珠粒附着到管的后壁，该溶液仍然是清楚的。除去用3毫升0.1％BSA / PBS两次径流和重复洗涤。 </li><li>重悬在4℃下在3毫升0.1％BSA / PBS的珠和存储，避光。 5天之内使用。 </li></ol></li><li>贴标CD146 + L-干细胞<ol><li>收获使用温和的非胰蛋白酶溶液用PBS漂洗后的间充质细胞。 注意:卷取决于培养瓶的大小（0.3毫升/厘米2介质或PBS）中。 <ol><li>除去培养基并用无菌PBS冲洗细胞三次。 </li><li>添加温和非胰蛋白酶溶液的适当体积（ 见表 1）。使用表 1中的溶液时，在孵化器在37℃下，直到细胞已经分离，约10分钟。 </li><li>通过加入L- MSC培养基，和离心机在500×g下5分钟使酶失活。 </li></ol></li><li>在0.1％BSA / PBS上清，重悬细胞，并计数与血球或自动细胞计数器细胞。 注:0.1％的BSA的体积/ PBS取决于培养瓶的大小:瞄准5或10毫升0.1N％BSA / PBS。 </li><li>重复步骤2.2.2重悬在3ml 0.1％BSA / PBS（块未细胞的专用量特异性结合位点，并保持细胞存活）。保持细胞在冰上。 </li><li>准备3毫升圆底管与主抗CD146抗体，并保持在冰上。 注意:被添加到细胞中的专用量初级抗体的浓度取决于所使用的抗体。对建议的抗大鼠CD146抗体见表 1。 </li><li>从步骤2.2.3添加的细胞的总体积到管与初级抗体。附近以15rpm，4℃紧紧孵育上的旋转样品混合器30分钟。 </li><li>离心细胞，在500 xg离心，4℃5分钟，取出3毫升0.1％BSA / PBS上清，重悬细胞沉淀。重复离心步骤。 </li><li>重悬在含有按照2.1节中的说明制备并以15rpm，4℃下在旋转样品混合器孵育30分钟的第二抗体包被的珠子3 ml溶液的细胞。 </li></ol></li><li>选择CD146 + L-干细胞<ol><li>将装有磁铁的标记CD146 + L间充质干管。阳性细胞将被吸引到管子的背面。无需移动管或触摸珠，收获与阴性细胞上清液，用无菌巴斯德移液管（这些既可以收集或基于实验设计丢弃）慢。 </li><li>从磁体取出管和重悬细胞在3毫升10％的BSA / PBS中。重复步骤2.3.1中描述的选择过程。四次（Σ5选择步骤）。 </li><li>悬浮CD146 + L-的MSC在预热的L- MSC培养基并返回到磁铁。 </li><li>在一个适当大小的培养瓶（0.4毫升/厘米2）上清，悬浮在L-MSC培养基和板块。作为一个经验法则，板中的细胞大小相同培养瓶中从该细胞前珠选择解除。 CD146 + L-MSCs的数目，因此，铺板密度，将取决于源动物的年龄，物种和菌株。总是瞄准的〜5,000个细胞/ cm 2的接种密度。 注意:珠不会干扰细胞生长和将逐渐消失的细胞增殖。 </li></ol></li></ol> 3.冷冻细胞<ol><li>使用以下冷冻介质:60％pentastarch溶液（10％pentastarch在0.9％NaCl）中，20％的FBS，12.5％CPDA-1，2.5％的NaCl（0.9％），5％二甲基亚砜（DMSO）22。 注意:如果不是所有的成分都可用，含5％DMSO，30％FBS和65％αMEM一个解决方案是一个很好的选择。 </li><li>重悬的细胞以0.5×10 6个细胞/ ml，等分试样中的各1ml冷冻管并用冷冻集装箱冻结O / N在-80℃。 </li><li>转移到液氮贮罐的第二天。 </li></ol> 4.细胞解冻<ol><li>解冻细胞的小瓶在37℃水浴中5分钟。 </li><li>悬浮细胞在10ml预热的培养基，离心500 XG 5分钟，21℃。 </li><li>除去上清，悬浮于10毫升培养基中的细胞沉淀。在通风盖的细胞培养的种子细胞在0.2ml / cm 2的培养基中于5000个细胞/ cm 2的处理过的瓶（ 例如，15毫升的75cm 2的烧瓶中）。 </li><li>培养的细胞，在5％O 2，5％的CO 2，直至 80-90％汇合收获和/或实验性使用。过汇合将诱导分化。当5000个/ 平方厘米播种，L-干细胞将在培养3-4天达到80-90％汇合。 </li></ol>

The authors have nothing to disclose.

主要L-干细胞的分离和培养提供了一个机会，以更好地了解它们的功能和它们与其他细胞群在细胞水平上的互动，以及它们在肺的发育，健康和疾病的作用。这是特别重要的，因为在缺乏这些细胞的特定单个标记的使得它几乎不可能来研究这些细胞中的原位 。与所有主要的细胞群，应该记住，这些细胞更可能改变它们的性质较长它们保持在培养物19（在24中综述）。保持左?...

间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。在开发过程中，包括肺，间质发育是线索的重要来源，而居民干细胞是可能的候选人是在这个中心。此外，有证据表明居民的MSCs在成人疾病扰动，包括癌症1,2和纤维化3。这些居民组织干细胞研究中的主要障碍是缺乏这些细胞4的分离和鉴定明确的标记。干细胞抗原-1（SCA-1）在小鼠中作为用于各种组织的干细胞的标志物被鉴定，并且可用于L-的MSC 5的隔离，但不幸的是没有已知的其他物种6直向同源物。研究人员已经报道了各种不同的分离方法对L- MSC的从任肺组织或流体的分离。 - / CD45 - - / CD90 +细胞 7，CD31 - / CD45 - /上皮细胞粘附分子（EpCAM的） -这些从荧光激活细胞分选（FACS）为基础的方法选择用于CD31变化/的Sca-1 +细胞8，多药耐药转运ATP结合盒G（ABCG2）阳性细胞9或赫斯特33342染料外排10，塑料粘附11,12和迁移出组织糜13。 在这里所提出的方法的优点是数倍。通过使用温和酶消化和密度梯度14中，可以得到包含的MSC，但不包括上皮或内皮细胞的密度范围的所有细胞。随后塑料吸附步骤确保只有mesenchymal细胞粘附和留在文化，消除白细胞。最重要的是然而，CD146 +选择步骤允许消除成纤维细胞，因为这些细胞不表达CD146。细胞粘附分子，CD146的表达是积极与多能相关，并且因此是一个很好的标志物从间充质细胞群体15-19剔除成纤维细胞。这是通过使用CD90作为选择标记的优点，因为它不仅在干细胞，而且在lipofibroblasts 5,20表示。在这个协议中，我们已经明确选择磁珠选择，因为它是在细胞上温和，而且整个过程可在无菌条件下进行。相对于生长方法这种隔离方法的另一个重要优点是，它是比较快的，6-10天，而不是在一个月或更长时间的生长方法。初始分离后三到五天的间充质人口准备CD146 +塞莱<html> ction;后3到5天，CD146 +细胞准备用于实验或可以冷冻供以后使用。培养的下降时间，从而提高了电池的质量的MSC朝向延长体外培养 19成纤维细胞转分化。最后，由于该协议的性质，有可能通过简单地调节消化酶和孵育时间的选择选择合适的抗体，或甚至其他器官系统这种方法适用于其它物种。 这种隔离方法的详细方案在下面给出， 并在图1A和1B分别设置在隔离和CD146 +亚群的随后选择的示意图。此外，详细信息包含用于传代，冷冻和解冻这些细胞。 广告/ 53782 / 53782fig1.jpg"/&gt; 图1.肺间质细胞（A）和随后的CD146 +细胞的选择（B）的最小=分钟的隔离的示意图概览; EDTA =乙二胺四乙酸; 第二抗体=二级抗体; α-CD146抗体=初级抗CD146抗体; -ve细胞= CD146阴性细胞; +已经细胞= CD146阳性细胞。 <a href="https://www.jove.com/files/ftp_upload/53782/53782fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Neutral Protease</td><td>Worthington Biochemical Corporation</td><td>LS02104</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>Collagenase I</td><td>Worthington Biochemical Corporation</td><td>LS004196</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>DNAse I</td><td>Sigma-Aldrich</td><td>D5025</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>Pentobarbital sodium (Euthanyl)</td><td>Bimeda-MTC Animal Health Inc, Dublin, Ireland</td><td>-</td><td>Use 0.2 ml for rat pups between 20-30 g; larger animals may need more.</td></tr><tr><td>Dulbecco&amp;rsquo;s PBS + Sodium-pyruvate + Glucose</td><td>Life Technologies</td><td>14287-072</td><td>Very crucial to use this type of D-PBS, as the calcium and magnesium that are present in this D-PBS facilitate facilitate the enzymatic digestion</td></tr><tr><td>Ficoll-Paque PREMIUM (1.073 g/cm&lt;sup&gt;3&lt;/sup&gt;)</td><td>GE Healthcare</td><td>17-5446-52</td><td>Density gradient media. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a &gt;45&amp;deg; angle at very low speed, and that the subsequent centrifugation is done at 19 &amp;deg;C.</td></tr><tr><td>&amp;alpha;MEM</td><td>Sigma-Aldrich</td><td>M8042</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>200 mM L-Glutamine</td><td>Life Technologies</td><td>25030-164</td><td></td></tr><tr><td>100x Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-062</td><td>Penicillin/Streptomycin/Fungizone</td></tr><tr><td>M-280 Dynabeads</td><td>Life Technologies</td><td>11205D</td><td>Magnetic beads</td></tr><tr><td>biotinylated polyclonal rabbit-anti-mouse IgG</td><td>Dako, Agilent Technologies</td><td>E0464</td><td>Biotinylated secondary antibody that matches with the anti-rat CD146 antibody described below</td></tr><tr><td>DynaMag5-magnet</td><td>Life Technologies</td><td>12303D</td><td>Magnet that is recommended for use with Dynabeads. NOTE: magnet should be chosen based on the manufacturer&amp;rsquo;s instructions of the magnet beads of choice.</td></tr><tr><td>TrypLE express&amp;nbsp;</td><td>Life Technologies</td><td>12605-028</td><td>Gentle non-trypsin alternative, use 1 ml of TrypLE express/25 cm&lt;sup&gt;2&lt;/sup&gt; surface area. After detachment, 10 ml L-MSC culture medium is sufficient to inactivate 3 ml TrypLE</td></tr><tr><td>anti-rat CD146 antibody</td><td>Lifespan Biosciences Inc.</td><td>C35841</td><td>12.5 &amp;mu;l per 0.5 x 10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Pentaspan (pentastarch solution)</td><td>Bristol-Myers Squibb Canada</td><td>-</td><td>Can be obtained through local blood donation services or hematology departments. Alternatively, one could use the PSI 20% Pentastarch solution (Preservation Solutions, PST001) diluted 1:1 with 0.9% NaCl.</td></tr><tr><td>Mr. Frosty freezing container</td><td>ThermoFisher Scientific</td><td>5100-0001</td><td>Improves viability when freezing L-MSCs overnight at -80 &amp;deg;C</td></tr></tbody></table>

isolation of cd146+ resident lung mesenchymal stromal cells from rat lungs

间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。此外，它们可能在正常器官发育的作用，并已被归因于疾病的作用，特别是那些患有纤维变性性质。这些居民组织干细胞研究中的主要障碍是缺乏对这些细胞的分离和鉴定明确的标记。这里所描述的隔离技术应用于肺间充质干居民（L-MSCS）的多个特征。一旦老鼠的牺牲，肺被删除，漂洗多次，以去除血。继手术刀机械分离，肺部使用I型胶原酶，中性蛋白酶和DNA酶I型的obtaine混合消化2-3小时D单细胞悬浮液，随后洗涤并铺在密度梯度介质（密度1.073克/毫升）中。离心后，从相间洗涤细胞并涂布在培养处理的烧瓶中。细胞是4-7天在生理5％O 2，5％CO 2的条件下培养。耗尽成纤维细胞（CD146 - ），并确保只有L-干细胞（CD146 +），CD146为+细胞阳性选择的人口通过磁珠选择执行。总之，这个过程可靠地产生主L-MSC的进一步的体外研究和操纵的群体。因为协议的性质，它可以很容易地转换为其他实验动物模型。

间充质干的，密度和贴壁的最可靠的物理特性二，在这个协议的第一部分是用来获取包含L-干细胞肺的间质细胞部分。虽然密度梯度相间将包括除了肺间质细胞的单核细胞和巨噬细胞，塑料粘附然后培养3-5天确保只有肺间质细胞保持。确实该细胞群表达了经典的MSC表面标记CD73，CD90和CD146，并为负标记CD34，CD45，主要组织相容性复合物II类（MHCII）-RT1B，CD11b和CD79A，表明不再?...

Watch this Scientific Journal Video about Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs at JoVE.com

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

这个协议描述了从大鼠获得原发性肺癌驻地间充质基质细胞，通过使用酶消化，密度梯度分离，塑料吸附和CD146 +磁珠选择的隔离技术。

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

isolation-cd146-resident-lung-mesenchymal-stromal-cells-from-rat

Research

JoVE Journal

Biology

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

8.3K 次观看.  Ottawa Hospital Research Institute. 这个协议描述了从大鼠获得原发性肺癌驻地间充质基质细胞，通过使用酶消化，密度梯度分离，塑料吸附和CD146 +磁珠选择的隔离技术。 

视频: Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

生物学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Primary Culture of Rat Hepatic Cells

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

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

大鼠肝细胞的分离和小学文化

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

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

小鼠原代肺内皮细胞的分离

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

<meta charset="utf8"></head><body>从小鼠肺中分离免疫和非免疫细胞，用于流式细胞分析

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>从小鼠肺中分离活髓细胞和上皮细胞群

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

从小鼠肺中分离活髓细胞和上皮细胞群

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

大鼠颌骨髓间充质干细胞的基于生态位的分离

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

作者聚焦：高纯度颚骨骨髓间充质干细胞在组织工程中的应用

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

大鼠颌骨骨髓间充质干细胞的分离和培养技术

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

CD146的分离

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此视频中的章节

小鼠肺树突状细胞的分离

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

对赫斯特的分离与表征^低 CD45^负小鼠肺间质干细胞

大鼠肝细胞的分离和小学文化

小鼠原代肺内皮细胞的分离

从小鼠肺中分离活髓细胞和上皮细胞群

从小鼠肺中分离活髓细胞和上皮细胞群

从小鼠肺中分离活髓细胞和上皮细胞群

大鼠颌骨骨髓间充质干细胞的分离和培养技术