这项研究得到了瑞士国家科学基金会 (SNSF, 格兰特 No. 320030_156193) 的支持。作者感谢 Dr. Benoît Werlen 提供 Biosilon 载体珠。我们还感谢 Prof. 和 Prof. 安德烈 Haeberli 帮助建立载体珠模型。

德国长白猪 (野生型养殖在当地的农舍和转基因育种研究所的分子动物繁殖和生物技术学院, 德国慕尼黑, 路德维希-马西米兰大学, 重量介于30公斤到40公斤之间, 被用于这项研究。所有的动物都被安置在标准条件下的水和食物 ad lib 。所有动物实验都是按照《英国动物法》 (科学程序) 和《 NIH 指南》对实验动物的照顾和使用以及瑞士动物保护法进行的。所有动物研究符合到达的指南。县兽医服务的动物实验委员会 (瑞士伯尔尼的小行政区) 批准了所有动物规程, 允许没有。BE70/14实验规程根据3R 原则被提炼, 并且最先进的麻醉和痛苦管理被用于减少动物的数量和减少动物的曝光在实验期间的重音和痛苦. 
 血液是根据瑞士司法管辖和伯尔尼大学医院的道德准则, 通过闭合系统静脉穿刺从健康人身上抽取的。词组和 #34; non-anticoagulated 血液和 #34; 意味着血液没有被抗凝治疗. 
 以下步骤在无菌条件下执行。熟悉碱性细胞培养无菌技术是必需的. 
 1. PAEC 的隔离 注意: 6 至10厘米的胸主动脉段是从3至6月的安乐死的德国长白猪获得的 (用于其他 体内 实验), 并立即转移到一个500毫升的玻璃瓶含有运输介质 (DMEM + 1% 青霉素/链霉素). 
<ol> <li> 预涂一个6井板, 纤维连接蛋白12.5 和 #181; PBS 1x 中的 g/毫升, 并把它放在一个孵化器在37和 #176; C 为 1 h. </li>
	<li> 预热无菌 PBS 1x 和细胞培养基 (DMEM). </li>
	<li> 从传输介质中取出猪主动脉. </li>
	<li> 将主动脉置于聚苯乙烯板上. </li>
	<li> 事先轻轻地用暖 PBS 冲洗. </li>
	<li> 纵向切开主动脉并用针固定. </li>
	<li> 在内容器表面添加暖细胞培养基. </li>
	<li> 吸入纤维连接蛋白-细胞培养基和添加新鲜细胞培养基 (DMEM 补充 10% FBS, 1% 青霉素/链霉素和0.4% 的内皮细胞生长培养基补充剂). </li>
	<li> 在细胞培养基中浸泡一棉签。在内容器表面的顶端轻轻地和缓慢地在同一方向上擦拭棉毛芽. </li>
	<li> 将6个井板中的单元格旋转一圈. </li>
	<li> 对其余的油井也这样做. </li>
	<li> 检查显微镜下的细胞, 将6井板放在孵化箱中37和 #176; C, 5% CO 2 . </li>
	<li> 在第二天更改媒体, 每 2-3 天更改一次. </li>
	<li> 当细胞要汇合时, 处理细胞并将它们种子成 T75 烧瓶 (PAEC P1). </li>
</ol> 2。PAEC 特性 <ol> <li> 前涂层 8 chamberslide 与纤维连接蛋白12.5 和 #181; PBS 1x 中的 g/毫升, 并把它放在一个孵化器在37和 #176; C 为 1 h. </li>
	<li> 种子 5 x 10 4 细胞/井和孵化过夜在孵化器在37和 #176; C. </li>
	<li> 使用 pbs 清洗两次电池 + + (pbs 补充 CaCl 2 和氯化镁 2 ), 300 和 #956; L/好。
	<li> 在室温、200和 #956 中用3.7% 甲醛固定电池10分钟; L/好. </li>
	<li> 使用 PBS 清洗电池3次 + + , 300 和 #956; L/好. </li>
	<li> 添加300和 #956; PBS 1 x-3% BSA (阻塞缓冲器), 室温下保持30分钟. </li>
	<li> 使用 PBS 1 x-1%BSA-0.05% 洗涤剂, 160 和 #956 中稀释的初级抗体 (抗 VE 钙黏附素, anti-CD31, anti-vWF); L/井, 室温下孵育1小时. </li>
	<li> 使用 PBS 洗涤3次 + + (200 和 #956; 我/好). </li>
	<li> 在 PBS 1 x 1%BSA-0.05% 洗涤剂、160和 #956 中应用二次抗体和 DAPI 稀释剂, 在室温下孵育1小时. </li>
	<li> 使用 PBS 洗涤3次 + + (200 和 #956; 我/好). </li>
	<li> 用甘油为基础的安装介质安装幻灯片, 并在荧光显微镜下验证内皮细胞标志物的表达. 
	注: 培养猪主动脉内皮细胞在 T175 烧瓶 (DMEM 低葡萄糖培养基 + 10% FBS, 1% 青霉素/链霉素和0.4% 的内皮细胞生长培养基补充剂) 直到90% 汇合到达。(播种密度 1 x 10 6 单元格, 90% 汇合大约对应于 5 x 10 6 单元格)。
</ol> 3。载体珠的涂层 <ol> <li> 将7毫升载体珠与42毫升的胶原蛋白溶液混合在一个50毫升的管中 (100 和 #956; 在0.2% 醋酸溶液中稀释), 在室温下孵育 1 h. </li>
	<li> 用25毫升 pbs pH 7.4 洗涤珠两次 (添加25毫升 pbs, 与吸管混合, 并等待, 直到珠被安顿下来, 然后丢弃上清和重复), 一次与25毫升的 DMEM 介质. </li>
	<li> 覆盖50毫升管中的珠子, 10mL 培养基199补充 10% FBS, 1% 青霉素/链霉素, 1% l-谷氨酰胺, 0.4% 的内皮细胞生长培养基补充剂和25和 #956; 肝素 (5000-毫升) 的 l, 并允许平衡10分钟在进一步使用之前. </li>
</ol> 4。收集单元格 <ol> <li> 从包含 PAEC 的 T175 烧瓶中删除细胞培养基, 并添加5毫升的 PBS pH 值 7.4. </li>
	<li> 从 T175 烧瓶中删除 PBS. </li>
	<li> 添加5毫升的 Trypsin-0.05%edta 和孵育 3-4 分钟, 在37和 #176; C. </li>
	<li> 通过用15毫升的细胞培养基冲洗烧瓶来收集细胞, 并在50毫升的管子中转移悬浮液. </li>
	<li> 在室温下离心 1200 x g 的细胞8分钟, 去除多余的培养基, 在5毫升的细胞培养基中重颗粒. </li>
</ol> 5。将细胞播种到搅拌器烧瓶中 <ol> <li> 将20毫升的细胞培养基添加到细胞悬浮和重. </li>
	<li> 将20毫升细胞培养基 (w/o 细胞) 加入到500毫升的磁力搅拌器烧瓶中. </li>
	<li> 将单元格添加到步骤3.3 中的洗涤载体珠, 并将其与25毫升的血清吸管进行仔细混合. </li>
	<li> 将珠子/电池混合物转到磁纺纱瓶中. </li>
	<li> 冲洗50毫升管与10毫升的细胞培养基收集剩余的细胞. </li>
	<li> 将额外的85毫升细胞培养基添加到微调器烧瓶中, 并在37和 #176 过夜放入恒温箱中; C 在振动筛上 (100 x g, 混合间隔: 3 分钟每45分钟). </li>
	<li> 添加50毫升的细胞培养基 (总体积200毫升), 并继续搅拌额外的24小时, 在37和 #176; C 在一个振动筛 (100 x g, 混合间隔: 3 分钟每45分钟). </li>
	<li> 添加无色 RPMI 培养基 (补充 10% FBS, 1% 青霉素/链霉素, 1% l-谷氨酰胺, 0.4% 的内皮细胞生长培养基补充剂和25和 #956; l 肝素) 直到320毫升的总体积达到. </li>
	<li> 每48小时更换培养基: 去除100毫升的旧培养基, 加入100毫升的新鲜补充无色 RPMI. </li>
	<li> 将单元格的区域性培养为5到7天。时间取决于细胞包裹的珠子的汇合状态. </li>
</ol> p 类 = "jove_title ">> 6。汇流验证 <ol> <li> 收集200和 #956; 使用吸管将细胞包裹的珠子移到聚丙烯管中. </li>
	<li> 3 次用600和 #956 清洗珠子; pbs 1x (添加 pbs, 倾斜管和轻轻混合, 以避免细胞脱落, 等待珠子安顿下来, 丢弃 pbs 和重复). </li>
	<li> 通过添加200和 #956 来固定10分钟的珠子; parapicric 酸的 L. </li>
	<li> 使用600和 #956 清洗3次; PBS 1x. </li>
	<li> 添加 DAPI 稀释在 PBS 1x 和孵化10分钟. </li>
	<li> 在玻璃滑梯上传送珠子, 用甘油为基础的安装介质应用片. </li>
	<li> 在共聚焦显微镜下可视化珠子. </li>
</ol> 7。实验过程 <ol> <li> 从磁性搅拌器烧瓶中取出电池包覆的珠子 (程序不必在无菌条件下进行), 用10毫升的血清学吸管将它们转移到12毫升的圆底聚丙烯管中. </li>
	<li> 让珠子安定下来 (大约 1-2 分钟) 并且去除多余的媒介. </li>
	<li> 添加更多的珠子到管, 直到每管包含整整2毫升的珠子. </li>
	<li> 在每根管子上加5毫升的透明 RPMI, 并使用10毫升的血清吸管进行仔细的混合. </li>
	<li> 让珠子沉淀下来, 去除多余的介质. </li>
	<li> 再重复一次洗涤过程与 RPMI 和去除所有多余的媒介. </li>
</ol> 8。非抗凝血的孵育 <ol> <li> 小心而缓慢地 (既不使用喷气机也不 vacutainers) 从一个健康的志愿者那里抽取血液并在9毫升的中性聚丙烯管中收集 (没有抗凝剂). </li>
	<li> 缓慢转移8毫升血液与10毫升血清学吸管到每一个聚丙烯管含有2毫升的细胞包覆珠 (总体积将是10毫升)。避免粗暴处理血液或珠子, 以避免过早的 EC 活化。该过程需要 1-2 min. </li>
	<li> 小心地倾斜血/珠混合物, 以确保相同的混合和密封的上限与石蜡膜. </li>
	<li> 将管子放在一个水平的倾摆台上 (仅有温和的倾斜设置) 在37和 #176 内; C 恒温箱和记录凝固时间。
	<ol> <li> 在设置的时间间隔内, 例如 在10、20、30、50、70、90分钟后, 删除至少 1.5-2 毫升的血珠混合物, 用于血清或血浆分析. 
		注: 对于6时间点, 我们建议在一组细胞内有超过3复制, 因为血液取样将在不同的管中进行. </li>
		<li> 收集血清, 让血液凝固。要收集血浆, 加入 EDTA 或柠檬酸盐到2毫升管前添加血液样本. </li>
		<li> 将管在冰上, 离心机在 2500 x g 为10分钟在4和 #176; c 和储存血清/血浆在80和 #176; c 直到使用. 
		注意: 材料的详细信息在 材料表 </li> </ol> </li> </ol> 中提供

<ol>
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作者没有什么可透露的。

该模型适用于凝血相关研究, 可以分析凝血的不同方面及其与 EC 的相互作用。8在异种移植的研究中, 用人血9对不同基因修饰的猪 ECs 的抗凝血性能进行检测是一个有用的系统。
该协议最关键的步骤是确保微在开始实验之前的完整的细胞覆盖率 (70-100), 以及那些关于仔细收集和处理 non-anticoagulated 全血以避免过早血小板活化由于高剪切应力, ?...

血管内皮由一单层内皮细胞 (EC) 组成, 它能使血管的腔管排成一线。在生理状态下, 静止 EC 负责维持一个抗凝和抗炎环境。1这是由 EC 表面的抗凝和抗炎蛋白的表达所介导的。例如, 由异移植器官的缺血/再灌注损伤或血管排斥引起的 EC 活化导致内皮表面从抗凝和抗炎状态转变为 pro-coagulant 和炎国家.1
为了研究内皮和凝血因子之间的奇妙和复杂的相互作用,在体外模型, 模仿尽可能密切的在体内情况是非常可取的。传统的体外凝血分析的一个共同的局限是使用抗凝血液, 使得凝血介导的作用复柠檬酸全血不能重现结果可获得新鲜 non-anticoagulated 血。2此外, 在传统的平板细胞培养系统中, 不可能利用内皮的抗凝血特性, 因为不能达到每个血液体积的足够的内皮细胞表面。该模型通过在球形载体珠表面培养 ec, 克服了这些局限性, 使 ec surface-to-blood 比 #62; 16 厘米2/毫升可以达到, 这是类似的情况下小动脉或静脉,被描述为足以使血液在 EC 表面进行 "自然" 抗凝。3,4全血可以在不添加抗凝剂的情况下使用。血液标本可在实验过程中采集, 细胞因子、凝血因子和可溶性补体活化标志物均可检测和量化。此外, 还可以通过共聚焦显微镜对补体和免疫球蛋白的沉积以及 ec 活化标记物的表达进行分析, 对 ec 包覆的载体珠进行研究。另一个有趣的应用包括测试药物, 这应该是防止内皮细胞活化, 从而, 凝血。5虽然这个模型不能完全取代动物实验, 它提供了一种方法来测试特定的功能假说,前体使用细胞, 从而减少了动物的数量, 用于基础研究的缺血/再灌注损伤或 (异) 移植。
用该模型模拟了猪主动脉内皮细胞 (PAEC) 在载体念珠上生长并与整个 non-anticoagulated 人血培养的异种移植环境。不同的转基因 PAEC, 携带了一些人类基因, 如 CD46 调节的补充系统和/或血栓调节蛋白 (hTBM) 的调节凝血系统, 分析了他们的抗凝血性能。内皮细胞活化、补体和凝血系统受到严密控制和相互连接。6因此, 重要的是要了解不同的转基因细胞在接触人体血液后的行为与黏附分子的表达和细胞因子的释放、糖的脱落以及抗凝蛋白的丧失有关。7

<table>
	<tbody>
		<tr>
			<td>PAEC</td>
			<td></td>
			<td></td>
			<td>Isolated from pig aorta in our Lab</td>
		</tr>
		<tr>
			<td>Microcarrier beads (Biosilon)</td>
			<td>Thermo Fisher Scientific</td>
			<td>160-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, Bovine</td>
			<td>Gibco</td>
			<td>A10644-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M7528</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>32404-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral tubes</td>
			<td>Sarstedt</td>
			<td>02.1726.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>Simport</td>
			<td>T406-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirrer flasks</td>
			<td>Tecnomara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological stirrer</td>
			<td>Brouwer</td>
			<td>MCS-104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti CD31</td>
			<td>R&#38;D Systems</td>
			<td>MAB33871</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG-Cy3</td>
			<td>Jackson Immuno Research</td>
			<td>112-166-003</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti VE-cadherin</td>
			<td>Santa Cruz</td>
			<td>sc-6458</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti vWF</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Dk anti Gt IgG &#8211; Alexa 488</td>
			<td>Molecular Probes</td>
			<td>A11055</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti Rabbit - FITC</td>
			<td>Southern Biotech</td>
			<td>4050-02</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>32670-25MG-F</td>
			<td>Dilution 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Concentration: 10% in cell culture medium</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton Dickinson</td>
			<td>367286</td>
			<td>Size: 0.8x19 mm (21&#610; 3/4&#34;)</td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Sarstedt</td>
			<td>14.1205</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 710</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Image J</td>
			<td></td>
			<td>Available for free at: https://imagej.net</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium SupplementMix</td>
			<td>PromoCell</td>
			<td>C-39216</td>
			<td>2mL in 500mL of DMEM</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>Heparinun natricum</td>
			<td>Drossa Pharm AG</td>
			<td></td>
			<td>Stock concentration: 5000 U.I./ mL</td>
		</tr>
		<tr>
			<td>Chamberslides</td>
			<td>Bioswisstec AG</td>
			<td>30108</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 x 2H2O</td>
			<td>Merck</td>
			<td>2382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x6 H2O</td>
			<td>Merck</td>
			<td>1.5833.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Axon Lab AG</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycergel mounting medium</td>
			<td>Dako</td>
			<td>C0563</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood

在体内, 内皮细胞是血液循环的自然抗凝血的关键。因此, 内皮细胞活化导致凝血。这种现象在许多临床情况下被观察, 象器官移植在存在预先 anti-donor 抗体, 包括异种移植, 并且在缺血或再灌注损伤。为了减少动物实验根据3R 标准 (减少, 替换和提炼),在体外模型研究内皮细胞活化作用对凝血是非常可取的。然而, 普通的平板系统的内皮细胞培养提供了一个表面积的比例为 1-5 厘米2的内皮每毫升的血液, 这是不足够的自然, 内皮介导的抗凝。在载体珠上培养内皮细胞可使表面积率提高到 40-160 cm2/毫升。这种增加的比例足以确保 "自然" 的全血抗凝, 从而可以避免使用抗凝剂。本文介绍了一种体外microcarrier-based 系统, 研究了猪内皮细胞的遗传修饰对全 non-anticoagulated 人血液凝固的影响。在所述的测定方法中, 原发猪主动脉内皮细胞, 无论是野生型或转基因的人 CD46 和血栓调节蛋白, 都生长在载体珠上, 然后暴露在新绘制的 non-anticoagulated 人血中。该模型可以测量和量化的细胞因子释放, 以及活化标志的补充和凝聚在血浆中。此外, 通过共聚焦显微镜对活化的内皮细胞进行成像, 并在 endothelialized 珠上进行免疫球蛋白、补体和凝血蛋白的沉积。这种化验也可以用来测试药物, 这是应该防止内皮细胞活化, 从而, 凝血。在它的潜力, 以减少动物的数量用于此类调查, 所述的化验是容易执行和一贯重现性。

在 7-10 天的文化在微调瓶 (图 1) 的细胞是汇合的, 覆盖整个表面的载体珠 (图 2)。70-100 状态的验证是一个重要的步骤, 因为 non-confluent 单层的 EC 在微将导致明显减少凝血时间, 因为载体珠的表面强烈 pro-coagulant (凝固时间: 4 ± 1 min) (图 3)。
另一个需要注意的重要...

Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

我们提出了一个体外模型, 允许对整个 non-anticoagulated 血液中的凝血进行研究和分析。该系统的抗凝血依赖于健康的内皮细胞的天然抗凝作用, 内皮细胞活化会导致血栓形成。

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

assessment-anticoagulant-anti-inflammatory-properties-endothelial

Research

JoVE Journal

Medicine

8.3K 次观看.  University of Bern. 我们提出了一个体外模型, 允许对整个 non-anticoagulated 血液中的凝血进行研究和分析。该系统的抗凝血依赖于健康的内皮细胞的天然抗凝作用, 内皮细胞活化会导致血栓形成。

视频: Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

科研

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

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

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

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 ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.
Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.
Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

血管内皮功能的超声评估:血流介导的舒张试验的技术指导原则

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...