Name: investigating aortic valve calcification via isolation and culture of t lymphocytes using feeder cells from irradiated buffy coat
Uploaded: 2021-02-04T13:30:00
Description: Watch this Scientific Journal Video about Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat at JoVE.com

由于Peter Rosenthal博士，Dirk Böhmer博士和Charité Benjamin Franklin放射科的整个团队的可用性，用于该协议的所有buffy外套袋都经过了辐照。奖学金获得者/Mary Roxana Christopher，这项工作得到了德国心脏学会（DGK）的奖学金支持。

这项研究是根据《确保良好科学做法慈善法》进行的，并遵守了关于隐私和道德的法律准则和规定。伦理委员会批准了所有人体实验，并根据道德表上报告的规则维护了患者的隐私和匿名性。
注：对于下面描述的方案，使用新鲜的人狭窄瓣膜样品。
1. 试剂制备
<ol><li>通过加入RPMI 1640培养基制备富集的RPMI完全培养基：非必需氨基酸;丙酮酸钠，L-谷氨酰胺...

<ol>
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D., 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=The+Lymphocytic+Infiltration+in+Calcific+Aortic+Stenosis+Predominantly+Consists+of+Clonally+Expanded+T+Cells.">The Lymphocytic Infiltration in Calcific Aortic Stenosis Predominantly Consists of Clonally Expanded T Cells.</a> The Journal of Immunology. 178 (8), 5329-5339 (2007).</li><li>Raddatz, M. A., Madhur, M. S., Merryman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+immune+cells+in+calcific+aortic+valve+disease.">Adaptive immune cells in calcific aortic valve disease.</a> American Journal of Physiology-Heart and Circulatory Physiology. 317 (1), 141-155 (2009).</li><li>Galkina, E., 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=Lymphocyte+recruitment+into+the+aortic+wall+before+and+during+development+of+atherosclerosis+is+partially+L-selectin+dependent.">Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.</a> The Journal of Experimental Medicine. 203 (5), 1273-1282 (2006).</li><li>Poursaleh, A., 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+intimal+endothelial+cells+from+the+human+thoracic+aorta:+Study+protocol.">Isolation of intimal endothelial cells from the human thoracic aorta: Study protocol.</a> Medical journal of the Islamic Republic of Iran. 33, 51 (2019).</li><li>Yun, T. J., Lee, J. S., Shim, D., Choi, J. H., Cheong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Aortic+Dendritic+Cells+and+Lymphocytes+in+Atherosclerosis.">Isolation and Characterization of Aortic Dendritic Cells and Lymphocytes in Atherosclerosis.</a> Methodsin Molecular Biology. 11559, 419-437 (2017).</li><li>Adams, N. M., Grassmann, S., Sun, J. 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在这里，我们提出了一种使用流式细胞术从狭窄主动脉瓣样品中分离的T淋巴细胞亚群的方法。这种方法需要使用辐照的黄褐色涂层来隔离PBMC。黄褐色外套袋必须承受的辐射频率为9000 Rad / 90 Gray（Gy），这是阻止饲养细胞增殖的关键步骤。从黄褐色外套袋中分离的细胞的作用是仅充当饲养细胞并为从瓣膜中分离的T细胞提供营养。正如过去25所指出的那样，使用单卷曲的巴菲外套袋...

钙化主动脉瓣疾病（CAVD）是最常见的心脏瓣膜疾病之一，对医疗保健有很大影响。在过去几年中，主动脉瓣置换术的频率急剧增加，并且由于老年人口不断增长，预计还会进一步增加1。
CAVD 的潜在病理生理学仅部分了解，目前的治疗策略仅限于保守措施或通过手术或经皮手术进行主动脉瓣置换术。迄今为止，除非进行主动脉瓣置换术 （AVR），否则没有有效的药物治疗可以阻碍或逆转 CAVD 的进展，并且高死亡率与早期症状发作相关2。在重度症状性主动脉瓣狭窄患者中，据报道，3 年无症状生存率低至 20%3。经导管主动脉瓣置换术（TAVR）代表了一种新的选择，彻底改变了高危患者的治疗，特别是在老年人中，并大大降低了死亡率，死亡率在该人群中本质上是很高的4，5，6。尽管TAVR取得了有希望的结果，但还需要进一步的研究来了解CAVD病理生理学，以确定新的早期治疗靶点7，8，9。
CAVD以前被认为是一种被动的退行性过程，现在被认为是一种主动进行性疾病，其特征在于主动脉瓣间质细胞的成骨细胞表型切换10。这种疾病涉及进行性矿化，纤维钙化变化和主动脉瓣运动减弱（硬化症），最终阻碍血流导致主动脉瓣开口变窄（狭窄）11。
炎症被认为是CAVD病理生理学的关键过程，类似于血管动脉粥样硬化的过程。内皮损伤可导致脂质种类的沉积和积累，尤其是主动脉瓣中的氧化脂蛋白12。这些氧化的脂蛋白引起炎症反应，因为它们具有细胞毒性，炎症活性导致矿化。最近强调了先天免疫和适应性免疫在CAVD发展和疾病进展中的作用13。在 CAVD 和矿化主动脉瓣小叶患者中，已经记录了记忆 T 细胞特定亚群的激活和克隆扩增，因此炎症过程被认为至少参与 CAVD 的发展，并且可能也参与疾病进展14。事实上，尽管健康瓣膜和患病瓣膜中都存在抗原呈递细胞和巨噬细胞，但 T 淋巴细胞的存在表明主动脉瓣老化和患病。这种淋巴细胞浸润以及新生血管形成和化生的增加是CAVD15的特征性组织学体征。
我们假设主动脉瓣间质细胞与免疫系统激活之间存在相互作用，这可能触发主动脉瓣中慢性炎症过程的开始。从狭窄的主动脉瓣组织中测定T细胞可能是阐明它们在钙化发展中的作用的有效方法，因为它可以提供体内主动脉瓣细胞的密切表示。在目前的工作中，使用主动脉瓣组织，我们分离T淋巴细胞，培养和克隆它们，然后使用荧光激活细胞分选（FACS）表征它们。从因严重主动脉瓣狭窄而接受手术瓣膜置换术的CAVD患者中切除新鲜主动脉瓣样本。手术切除后，将新鲜瓣膜样品小块解剖，培养T细胞，克隆，然后使用流式细胞术进行分析。染色过程简单，染色管可以使用0.5%的多聚甲醛固定，并在15天后进行分析。从染色面板生成的数据可用于跟踪与干预相关的T淋巴细胞分布随时间的变化，并且可以很容易地进一步开发以评估感兴趣的特定T细胞亚群的活化状态。
由于自发荧光等问题，钙化组织的提取，从钙化组织中分离白细胞，特别是在这种类型的组织上使用流式细胞术可能具有挑战性。很少有出版物具有用于此特定目的的协议16，17，18。本文介绍了一种专门设计用于从人主动脉瓣样本中直接分离和培养T淋巴细胞的方案。淋巴细胞克隆扩增是适应性免疫的标志。在体外研究这一过程提供了有关淋巴细胞异质性水平的有见地的信息19。经过三周的潜伏期后，T细胞克隆准备被种植，因为从每个克隆中获得了足够量的T细胞，以便进行表型和功能研究。随后通过细胞荧光测定法研究T克隆的表型。
该免疫方案是对Amedei等人先前开发的用于从人体组织中分离和表征T细胞的方法的改编，专门设计用于钙化人体组织，例如在CAVD20，21，22中。这里使用辐照的矮黄色外套分离PBMC（外周血单核细胞）的方案描述了获得饲养层细胞（FC）的有效方法，该方法专门针对从瓣膜间质细胞中分离的T淋巴细胞的克隆阶段进行调整。饲养层由生长停滞的细胞组成，这些细胞仍然具有活力和生物活性。饲养细胞的作用对于支持从瓣膜间质细胞中分离的T淋巴细胞的体外存活和生长非常重要23。为了避免饲养层细胞在培养物中增殖，这些细胞必须经历生长停滞。这可以通过两种方式实现：通过物理方法（如辐照）或通过细胞毒性化学物质（如丝裂霉素C（MMC））进行处理，丝裂霉素C是一种可以直接应用于培养表面的抗肿瘤抗生素24。在这里，我们展示了通过细胞照射实现的饲养层细胞生长停滞。
该方法提供了一种从主动脉瓣组织中分离和表征T细胞的有效，经济有效的方法，有助于扩大用于探索CAVD病理生理学的免疫学方法的范围。

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		<tr>
			<td>50 mL plastic syringes</td>
			<td>Fisherbrand</td>
			<td>9000701</td>
			<td></td>
		</tr>
		<tr>
			<td>96- well U- bottom Multiwell plates</td>
			<td>Greiner Bio-One</td>
			<td>10638441</td>
			<td></td>
		</tr>
		<tr>
			<td>Bag Spike (needle free)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Dilute to 4% with PBS</td>
		</tr>
		<tr>
			<td>CD14 Brilliant violet 421</td>
			<td>&#160;Biolegend</td>
			<td>560349</td>
			<td></td>
		</tr>
		<tr>
			<td>CD25 PE</td>
			<td>&#160;Biolegend</td>
			<td>302621</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 PE/Cy7</td>
			<td>&#160;Biolegend</td>
			<td>300316</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 Alexa Fluor 488</td>
			<td>&#160;Biolegend</td>
			<td>317419</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Brilliant violet 711</td>
			<td>&#160;Biolegend</td>
			<td>304137</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8 Brilliant violet 510</td>
			<td>&#160;Biolegend</td>
			<td>301047</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube 1.5 mL</td>
			<td>Eppendorf</td>
			<td>13094697</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical centrifuge tube</td>
			<td>Falcon</td>
			<td>10136120</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>10788561</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polystyrene Tubes</td>
			<td>BD</td>
			<td>2300E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast read 102 plastic counting chamber</td>
			<td>KOVA INTERNATIONAL</td>
			<td>630-1893</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters for culture medium 250 mL</td>
			<td>NalgeneThermo Fisher Scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters for culture medium 500 mL</td>
			<td>NalgeneThermo Fisher Scientific</td>
			<td>166-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>HB 101 Lyophilized Supplement</td>
			<td>Irvine Scientific</td>
			<td>T151</td>
			<td></td>
		</tr>
		<tr>
			<td>HB Basal Medium</td>
			<td>Irvine Scientific</td>
			<td>T000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-Inactivated FBS (Fetal Bovine Serum)</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>HS (Human serum)</td>
			<td>Sigma Aldrich</td>
			<td>H3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2 IS</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-744</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids solution</td>
			<td>Sigma</td>
			<td>11082132001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>10538931</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>PHA (phytohemagglutinin)</td>
			<td>Stem Cell Technologies</td>
			<td>7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>Thermo Scientific</td>
			<td>R80115TS</td>
			<td>10 0mm x 15 mm</td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>HyClone</td>
			<td>15-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco by Life technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters 0,45&#181;l</td>
			<td>Rotilabo-Spritzenfilter</td>
			<td>P667.1</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 Cell culture flasks</td>
			<td>InvitrogenThermo Fisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Cell culture flask</td>
			<td>Thermo Fisher Scientific</td>
			<td>10232771</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;- Mercaptoethanol</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating aortic valve calcification via isolation and culture of t lymphocytes using feeder cells from irradiated buffy coat

钙化主动脉瓣疾病 （CAVD） 是一种活动性疾病过程，从瓣膜轻度增厚到严重钙化，尽管存在新的治疗选择，如经导管主动脉瓣置换术 （TAVR），但死亡率较高。 
从瓣膜钙化开始并导致严重主动脉瓣狭窄的完整途径仍然部分了解。通过提供体内主动脉瓣细胞的密切表示，从狭窄瓣膜组织中测定T淋巴细胞可能是阐明它们在钙化发展中的作用的有效方法。手术切除后，将新鲜的主动脉瓣样品小块解剖，培养T淋巴细胞，克隆，然后使用荧光活化细胞分选（FACS）进行分析。
染色过程简单，染色管也可以使用0.5%的多聚甲醛固定，并在15天后进行分析。从染色面板产生的结果可用于跟踪与干预相关的T细胞浓度随时间的变化，并且可以很容易地进一步开发以评估感兴趣的特定T细胞亚型的活化状态。在这项研究中，我们展示了在新鲜钙化主动脉瓣样品上进行的T细胞分离，以及使用流式细胞术分析T细胞克隆的步骤，以进一步了解适应性免疫在CAVD病理生理学中的作用。

我们使用一种简单且具有成本效益的方法表征来自人类严重主动脉瓣狭窄患者的新鲜主动脉瓣样本的白细胞群（参见方案）。分离PBMC的方法是获得饲养层细胞的重要步骤，饲养层细胞用于实验的每个步骤（克隆，再喂养和分裂阶段），并能够检测和表征主动脉瓣样品中浸润的白细胞。此方法的关键步骤如图 1 所示。
<img alt="Figure...

Watch this Scientific Journal Video about Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat at JoVE.com

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

在本研究中，我们描述了从钙化主动脉瓣新鲜样品中分离T淋巴细胞的过程，以及使用流式细胞术分析表征适应性白细胞亚群的T细胞克隆的分析步骤。

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

该协议易于执行。克隆阶段允许从新鲜样品中获得大量T克隆。此外，T细胞的表型和功能可以很容易地分析。演示该程序的将是玛丽·克里斯托弗（Mary Christopher），她是我实验室的博士生。要诱导T细胞克隆，请使用带有无针阀的袋尖刺打开层流罩中的辐照淡黄色外套袋。将多达50毫升的血液从袋子转移到T-150烧瓶中，并在烧瓶中加入70毫升PBS。小心地将30毫升稀释的血液分层到四个50毫升锥形管中每个20毫升的密度梯度上，并通过密度梯度离心分离细胞。小心地吸出上清液，并将PBMC从每个管转移到单个新的50毫升管中。使用PBS将最终体积提高到50毫升，并通过离心收集PBMC。在相同条件下进行第二次洗涤后，将分离的饲养细胞悬浮在10毫升细胞培养基中进行计数，并将25毫升70毫升细胞培养基加入两个50毫升管中。然后将饲养层细胞以2×10的2×10加入每毫升补充IL-2浓度的细胞培养基的第6个细胞中。为了建立T淋巴细胞培养物，将含有来自狭窄瓣膜样品瓶的上清液的T细胞克隆池入单个50毫升管中，并通过离心收集细胞。每次洗涤在50毫升PBS中洗涤沉淀三次，然后将细胞重新悬浮在1毫升细胞培养基中进行计数。将细胞稀释至每毫升浓度为1×10至第3个细胞，并将500微升细胞加入补充有IL-2的细胞培养基管中，不含饲养层细胞。将饲养池悬浮液加入塑料100×15毫升培养皿中，并向适当数量的U型底96孔板的每个孔中加入100微升的饲养池进行实验。然后将T细胞加入培养皿中，并向补充良好的每个饲养细胞中加入100微升T细胞，在细胞培养箱中孵育一周。PBMC分离对于获取饲养层细胞以及检测和表征主动脉瓣样品中浸润的白细胞至关重要。孵育两周后，可以获得克隆T细胞群。这里显示了用于分析钙化主动脉瓣疾病患者T细胞亚群的门控方案。正如在该患者中观察到的那样，CD4 + T细胞比CD8 + T细胞更多。未克隆 T 细胞的流式细胞术分析证实，天然瓣膜和克隆样品中都存在相同的 T 细胞标志物。综上所述，这些数据表明T细胞存在于具有CD45 +白细胞的钙化主动脉瓣中，这表明钙化主动脉瓣疾病与免疫系统的激活和炎症活性有关。这种方法非常通用。它可以应用于不同的方案，研究人员需要能够从组织中分离细胞，以进行功能表征。请记住，在无菌条件下工作以避免污染，并在显微镜下批判性地观察细胞非常重要。

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Isolation of Double Negative ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â² T Cells from the Kidney

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...