我们非常感谢博士的莱拉Shakeri博士阿雷夫Dournaei的关键铁饼和穆罕默德·礼萨·哈德姆谢里夫先生为他的技术支持。

1。准备的酶液和增殖培养基（PM） <ol><li> 车型胶原酶类型I解：称出I型胶原酶（12毫克/毫升），并溶解在1ml PBS及过滤器，用0.2μm注射器过滤器。然后将其放在15毫升的试管，并保持在-20℃，直到需要。 </li><li> 使分散酶解：称取分散酶（16毫克/毫升），并溶解在1毫升的PBS及过滤器，用0.2μm注射器过滤器。然后将其放在15毫升的试管，并保持在4℃直至需要。 </li><li> 酶溶液：加入到2毫升无菌PBS中含有100毫克/ ml青霉素，100毫克/ ml链霉素1毫升胶原酶I型溶液（12毫克/毫升）和1毫升分散酶解决方案（16毫克/毫升）。最终成交量为纯净和胶原酶我总含量应为4毫克/毫升和3毫克/毫升，容易接受。然后，分装到4个15毫升的试管，每片含1毫升酶溶液。此卷每个管可以用于一个纸浆的蒸解。 </li><li> 使清洗溶液：加入100毫克/ ml青霉素，100毫克/毫升链霉素的PBS中。 </li><li> 车型基本培养基：阿尔法修改的Eagle培养基（α-MEM），补充有10％FBS及100单位/ ml青霉素，100毫克/毫升链霉素。 </li><li> 设为的扩散介质（PM）：阿尔法修改的Eagle氏培养基（α-MEM）辅以10％胎牛血清，100μML-抗坏血酸-2 -磷酸，2 mM的L-谷氨酰胺，100单位/ ml青霉素，100毫克/毫升0.25毫克/毫升链霉素，两性霉素B。 </li><li> 车型牙源性媒体：阿尔法修改的Eagle氏培养基（α-MEM）补充有10％胎牛血清，100μML-抗坏血酸-2 -磷酸，2 mM的L-谷氨酰胺，100单位/ ml青霉素，100毫克/ ml链霉素，0.01μM地塞米松，5mM的β-甘油磷酸，1.8 mM的磷酸二氢钾。 </li></ol> 2。准备人牙髓组织中牙髓干细胞Isolatio的Ň <ol><li>正常的人类第三磨牙的成年人（21-29岁）下认可的机构审查委员会（IRB）的伊玛目阿里在牙科诊所。 </li><li>牙齿被置于基本培养基（α-MEM培养基补充有10％FBS），在4℃下，并转移到实验室</li><li>在无菌条件下，一个生物危害的层流罩内工作，设置被完成一个100毫米的培养皿中，以被处理的每个齿。齿面，用70％乙醇清洗。 </li><li>虽然工作在一个培养皿，牙齿保持的牙科镊子和一个手术刀，清洁碎片和牙周膜的根上。 </li><li>釉牙骨质交界处慢慢减少使用消毒的牙科盘揭示牙髓腔。它应该被认为必须缓慢进行，切割过程中的牙齿组织，以减少过热。 </li><li>轻轻分开的牙冠和牙根的牙髓组织。 </李&gt; <li>牙髓组织切成小块与援助的手术刀刀片。 </li></ol> 3。人牙髓隔离的<ol><li>牙髓组织进行的hDPSCs隔离的牙髓组织的外植体牙髓组织或细胞直接产物无论是酶解。 </li><li> 牙髓组织的酶分解： <ol><li>牙髓组织的小片的酶溶液在37℃下1小时，转移到涡，每30分钟到分手组织。 </li><li>后来被拆除，大细胞聚集和单细胞悬浮液，通过细胞通过一个70μM细胞的过滤器。 </li><li>单细胞悬浮液，在1,200 rpm下离心5分钟，在室温下。 </li><li>上清液仔细移液和沉淀物重新悬浮在1ml的增殖培养基（PM）的。 （ 注：FBS增殖培养基终止酶的显示解离）。 </li><li>单细胞悬浮液接种到25cm 2的培养瓶中用PM牙髓，然后在5％CO 2在37℃下孵育。 </li><li>培养基中被改变，直到细胞汇合实现每三天。 </li></ol></li><li> 直接从牙髓组织的外植体细胞的产物： <ol><li>牙髓组织切碎成1-2毫米的碎片，每片被放置在个25-cm 2培养瓶中用PM，然后温育在37℃下在5％CO 2。它应被视为细胞产物，总体积的PM必须支持进一步的细胞生长（2-3毫升按每烧瓶）的纸浆片的安装。 </li><li>换液后的产物观察。 </li><li>超越在汇合的细胞子的比率为1:4（通道1）培养。 </li></ol></li></ol> 4。免疫表<ol><li> 250,000细胞/管的这两种类型的细胞在通道3由PE或FITC标记的抗人用自己的同种型的抗体温育30分钟，于4℃下在黑暗的地方。 （ 注：施加的抗体浓度根据制造协议。） </li><li>的孵育时间后，加入100μlPBS或FACS稀释液加入到每个试管中，并在1,200 rpm下离心5分钟，在黑暗的地方。 </li><li>除去上清液与沉淀物重新悬浮于100μl的PBS或FACS稀释在黑暗的地方。 </li><li>有针对性的表面标志物（CD标记）BD FACSCalibur进行了评价。 </li></ol> 5。矿化细胞诱导分化的DPSCS量化的茜素红S含量<ol><li>牙髓组织任酶解（称为作为DPSC-ED）或直接的细胞生长自牙髓组织的外植体（DPSC-OG），通过以下方式获得的全部牙髓之中子培养第3代。 </li><li>一吨第3代，将细胞种子到6孔板中，以5,000细胞/孔。 </li><li>在60％的汇合，牙源性介质取代以往的PM。三个孔作为阴性对照，加入生长培养基中保持。 </li><li>换液，每3天至21天。 </li><li>在第21天，将细胞用PBS洗涤，1ml /孔，在室温下的15分钟的10％多聚甲醛固定。 </li><li>然后，多聚甲醛，用移液管关闭，仔细和细胞洗涤3次（每次5-10分钟）用蒸馏水，H 2 O。 （ 注：避免干扰单层。） </li><li>在洗涤后，1ml /孔茜素红S中加入在室温下20分钟。 </li><li>由去离子H 2 O 4次（每次5分钟，用温和的摇摆）洗涤细胞以除去任何额外的茜素红。 </li><li> 1ml /孔中加入H 2 O，以防止细胞干燥。 </li><li>板被假定为视觉插件pection和/或图像采集。 </li><li>茜素红S的定量测定对于由1毫升10％乙酸到6孔板与在室温下振摇孵育30分钟的各孔中，H 2 O取代。 </li><li>然后，单层细胞/孔分别为刮＆醋酸及细胞被转移到单独的1.5毫升微量离心管。 （ 注：每口井应该转移到一个单独的管即三个测试井和三个控制井为每个ED＆OG组。） </li><li>管被涡旋大力，持续30秒。 </li><li>加热至85℃，持续10分钟。 （ 注：为避免蒸发，管密封，封口膜）。 </li><li>加热结束后，将试管转入冰浴5 min。 （ 注 ：管不应该被打开，直至成为冷却）。 </li><li>浆料在20,000 xg离心15分钟，离心分离。在离心过程中，茜素红的标准了。 </li></ol>茜素红S的标准制备首先通过的稀释缓冲液稀释，及用于基于该表的2 - 倍系列稀释。 <table border="1"><tbody><tr><td colspan="7"> 高浓度范围内的染料 </td><td colspan="7"> 低浓度范围内的染料 </td></tr><tr><td> 2毫米</td><td> 1毫米</td><td> 500μM </td><td> 250μM </td><td> 125μM </td><td> 62.5μM </td><td> 31.3μM </td><td> 15.6μM </td><td> 7.8μM </td><td> 3.9μM </td><td> 1.9μM </td><td> 0.9μM </td><td> 0.4μM </td><td>空白</td></tr></tbody></table><ol start="16"><li>离心后，被转移到新的，独立的1.5毫升微量离心管1毫升上清液/管。 </li><li>将pH中和〜375微升10％氢氧化铵，按每管的。 （ 注：pH值的愤怒应该是4.1-4.5）。 </li><li>一个不透明的壁透明底96孔板中加入150μl的标准样品（包括单独的测试及控件）。 </li><li>读出在405nm处的吸光度，并从样品的OD值相比，标准样地，作进一步的分析。 </li></ol>

<ol>
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Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

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该协议描述了hDPSCs牙髓使用两种方法，酶解和牙髓组织的外植体干细胞的直接产物的分离和鉴定。此外， 在这些细胞的体外分化成成牙本质细胞，通过茜素红S的定量测定＆QPCR评估。 现有的协议，用于隔离从人类牙齿的牙髓组织已被用于各种仪器，如钳子（骨镊子）9，摘除手术中，针29，格雷西刮匙30，牙科裂缝毛刺4，等，这些方法是?...

干细胞是细胞克隆细胞具有两个显着的特点，被称为多潜能和自我更新15。在所有的干细胞不同的复制效力，牙齿干细胞的产后干细胞引起的关注，近年来，因为其可访问性，可塑性强，高增殖能力比较与其他成体干细胞16。典型地，类似的间充质干细胞，牙髓干细胞粘附的克隆形成有多个成间充质细胞的分化能力和/或非间充质细胞谱系，无论是在体外和体内的细胞，这些细胞。1-8,17,18稳干细胞鉴定阴性表达造血抗原（ 如 CD45，CD34，CD14），阳性表达的CD90，CD29，CD73，CD105，CD44和STRO-1。19,20 容易获得潜在的疼痛和发病率最低，作为一个有价值的人DPSCS能源间充质干细胞相比普通的来源，如骨髓间充质干细胞21。在一般情况下，稳干细胞已被分离由任何生长的方法， 即 ，从的牙髓组织外植体（DPSC-OG）22-24，和/或酶消化4,6,25（DPSC-ED）的干细胞的迁移。以往的研究表明，隔离方法和培养条件下，可以吸引不同的人群或谱系体外通道26,27。在的箱子恒牙（pDPSCs），Huang 等人透露，酶消化pDPSCs超越全部牙髓之中相比，有较高的增殖潜力，26此外，在箱子乳牙（dDPSCs），它表明一个STRO-1及CD34的标记表示在比较更多的dDPSC-ED与dDPSC-OG。此外，dDPSC-ED显示定义的骨/ odonto介质的高矿化率在27因此，由于出色的潜力DPSCS在regenerative药，将有更多的研究，以更好地了解可能来自不同的隔离方法不同人群的需要。 在这里，它是尝试引入纸浆提取的简单的方法，通过使用一步牙科金刚石盘以方便处理纸浆提取。此外，在申请ED或OG方法的人牙髓源性干细胞的分离，两组间的一般特性及分化能力进行了调查。

<table border="1"><tbody><tr><td>的试剂的名称</td><td>公司</td><td>目录或地段。数</td><td>评论（可选） </td></tr><tr><td>的α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> I型胶原酶</td><td> Sigma-Aldrich公司</td><td> C0130-100MG </td><td></td></tr><tr><td>中性蛋白酶</td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td>两性霉素B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td>定义胎牛血清（FBS） </td><td>胎牛血清</td><td> SH30070.03 </td><td></td></tr><tr><td> L-抗坏血酸-2 - 磷酸</td><td>西格玛</td><td> A8960-5G </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td>地塞米松</td><td>西格玛</td><td> D4902 </td><td></td></tr><tr><td> β-甘油磷酸二钠盐水合物，BioUltra </td><td>西格玛</td><td> G9422-100G </td><td></td></tr><tr><td>磷酸二氢钾</td><td> Sigma-Aldrich公司</td><td> P5655 </td><td></td></tr><tr><td>成骨检测试剂盒</td><td>密理博</td><td> PS01802031 </td><td></td></tr><tr><td>同型对照小鼠IgG2b-PE </td><td> BD Pharmingen公司</td><td> 50808088029 </td><td></td></tr><tr><td> FITC小鼠IgG2b同种型控制</td><td> BD Pharmingen公司</td><td> 559532 </td><td></td></tr><tr><td> FITC小鼠IgG1κ型</td><td> BD Pharmingen公司</td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE的小鼠抗人CD34/CD45 </td><td> BD Pharmingen公司</td><td>341071 </td><td></td></tr><tr><td> PE抗人CD146 </td><td> BD Pharmingen公司</td><td> 550315 </td><td></td></tr><tr><td>单克隆小鼠抗人CD90/FITC </td><td>特嘉</td><td> 00034418 </td><td></td></tr><tr><td> PE的小鼠抗人CD73 </td><td> BD Pharmingen公司</td><td> 550257 </td><td></td></tr><tr><td>抗H CD105/Endoglin PE的</td><td> BD Pharmingen公司</td><td> FAB10971P </td><td></td></tr><tr><td> PE的小鼠抗人CD11b/Mac1 </td><td> BD Pharmingen公司</td><td> 5553888 </td><td></td></tr><tr><td>小鼠抗人CD44 PE </td><td> BD Pharmingen公司</td><td> 555479 </td><td></td></tr><tr><td>磷酸盐缓冲溶液（PBS）中</td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70微米的细胞过滤器</td><td>鹰</td><td> 352360 </td><td></td></tr><tr><td&gt; 0.2微米针头式过滤器</td><td>新型Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25厘米2的培养瓶中</td><td> Sigma-Aldrich公司</td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> multiskan微孔板分光光度计</td><td> Thermo Scientific的</td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense显微镜</td><td>奥林巴斯</td><td></td><td></td></tr><tr><td>流动软件版本2.3.1 </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt;开发智齿容易访问的源干细胞在成年后可以得到常规正畸治疗。人牙髓干细胞（hDPSCs）具有高增殖能力，多系分化能力比较普通的成体干细胞来源<sup&gt; 1-8</sup因此，hDPSCs可能是自体移植，组织工程和再生医学的很好的候选人。除了这些优点，具有间充质干细胞（MSC）的功能，如immunolodulatory效果，使hDPSCs更有价值，即使在同种异体移植的情况下，<sup&gt; 6,9,10</sup&gt;。因此，使用此的干细胞来源的主要步骤，用于选择最佳的协议，用于隔离hDPSCs从牙髓组织。为了实现这一目标，关键是要研究不同的隔离条件下对不同细胞的行为，如他们共同的表面标记和其分化能力的影响。</p&gt;<p class="jove_content"因此，在这里我们分离的人牙髓组织影响第三磨牙，然后用现有文献的基础上协议，用于隔离hDPSCs，<sup&gt; 11-13</sup&gt;<em&gt; IE浏览器</em&gt;牙髓组织中的酶分解的（DPSC ED）或组织块长出（DPSC OG）。在这方面，我们努力促进的隔离方法，使用牙科金刚石盘。然后，将这些细胞其特征在于基质相关的标志物（CD73，CD90，C​​D105和CD44），造血/内皮标志物（CD34，CD45及CD11b）的，血管周围标记，如CD146和STRO-1。此后，这两个协议进行比较的基础上通过定量聚合酶链反应（QPCR）和茜素红染色成牙本质细胞的分化潜能。 QPCR被用于评估矿化相关基因的表达（碱性磷酸酶ALP，基质细胞外磷酸糖蛋白; MEPE牙本质涎磷蛋白; DSPP）。<sup&gt; 14</sup</p

<ol><li> 酶解（DPSC-ED）是通过以下方式获得的全部牙髓之中这里示出在第10天，15,18（图1）。发起的菌落上形成几乎为3至5天之后，隔离。 </li><li> 超越DPSCS（DPSC-OG），如图2所示。成纤维细胞样细胞开始从牙髓组织迁移到烧瓶平行订购了近5天播种后5天，10日，13日及18。 </li><li> 全部牙髓之中使用这两种方法在通道3的图3中所示，这两种类型的稳干细胞几乎相同的大小...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

通过使用所描述的方法分离和鉴定人牙髓干的细胞（hDPSCs）<strong酶解浆（DPSC-ED）</strong&gt;或<strong的直接产物牙髓组织的外植体干细胞（DPSC-OG）。接着</strong&gt;<em在体外</em这两种类型的hDPSCs&gt;比较分化成成牙本质细胞。

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

isolation-characterization-comparative-differentiation-human-dental

Research

JoVE Journal

Medicine

26.3K 次观看.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 通过使用所描述的方法分离和鉴定人牙髓干的细胞（hDPSCs）

视频: Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

科研

医学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

牙种植体不同材料的口腔生物膜形成

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

丙戊酸在人脐带血源性 CD34 + 细胞造血干细胞体外扩增中的作用

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...