这项工作得到了中国自然科学基金（82070450 to C.T.和 82170466 to L.Z.）和中国博士后科学基金（7121102223 to F.L.）的资助。

以下所有动物程序均由苏州大学机构动物护理和使用委员会批准。
1.试剂和材料准备
<ol><li>利用不含钙和镁的 1x PBS 和 2.5 U/mL 肝素钠盐制备灌注溶液。储存在4°C，使用时在冰上预冷。</li>
	<li>用HBSS稀释1250CDU/mL胶原酶II和3000U/mL DNase I，制备含有125 CDU/mL胶原酶II和60U/mL DNase I的解离试剂A。储存在4°C直至使用。</li>
	<li>通过将 1 mL 不含 EDTA 的胰蛋白酶（不含酚红）稀释到 1 mL 的 1x PBS 中，制备含有终浓度为 0.12% 胰蛋白酶的解离试剂 B。储存在4°C直至使用。</li>
	<li>在 1x PBS 中制备 10 mL 的 1.5% FBS 和 10 mL 的 5% FBS。使用1.5%FBS汇集颈动脉，并在使用前保持冰敷。使用5%FBS中和消化并在室温（RT）下储存。</li>
	<li>获取实验材料，包括 1 mL 注射器、20 mL 注射器和 26-G 针头、六孔细胞培养板、1.5 mL 离心管、40 μm 无菌细胞过滤器和冰容器。</li>
</ol>2、设备准备
<ol><li>对所有手术设备进行消毒，包括立体显微镜、解剖剪刀、显微解剖剪刀和细尖镊子。</li>
	<li>打开自动细胞计数仪并将其保持在室温。</li>
	<li>提前将水浴打开至37°C。</li>
	<li>使用前将微量离心机预冷至4°C。</li>
</ol>3.小鼠颈动脉的隔离
<ol><li>通过腹膜内注射（每10g体重0.24mL）使用1.25%三溴乙醇麻醉小鼠，并在3分钟后翻转鼠标以检查是否有矫正反射。然后，通过颈椎脱位对小鼠实施安乐死，并将其仰卧。用75%的酒精喷洒小鼠的皮肤。</li>
	<li>使用消毒的解剖剪刀打开皮肤并露出胸腔。切开隔膜。</li>
	<li>继续向上切割至下颌，小心地去除多余的结缔组织和脂肪，露出颈动脉。</li>
	<li>切开小鼠的下腔静脉，使血液从闭合循环中流出。</li>
	<li>用注射器缓慢连续地将 20 mL 预冷灌注溶液注入左心室。 注意：如果灌注成功，肝脏、脾脏和肾脏等富含血液的器官将变成灰白色。</li>
	<li>在立体显微镜下用显微解剖剪刀剥离颈动脉周围的所有脂肪和结缔组织。 注意：此步骤应缓慢而小心地进行，以防止颈动脉受损。</li>
	<li>从小鼠中分离颈动脉，用1x PBS洗涤以再次冲洗血液，并在冰上转移到含有1.5%FBS的六孔板中。</li>
</ol>4.将颈动脉消化成单细胞悬液
<ol><li>使用显微解剖剪刀纵向解剖颈动脉，并平放在另一个含有 1 mL 1.5% FBS 的 6 孔细胞培养板中。</li>
	<li>用 1.5% FBS 小心冲洗内膜以去除残留的血液。 注意：冲洗应轻柔缓慢，以免损伤内皮细胞。</li>
	<li>使用显微解剖剪刀在1.5%FBS中将每条颈动脉切成约2mm2 组织块。</li>
	<li>将这些组织块转移到具有1mL宽孔尖端的1.5mL离心管中，在4°C下以400× g 离心5分钟以将组织沉入底部。</li>
	<li>弃去上清液并将组织重悬于500μL解离试剂A中。</li>
	<li>将试管置于37°C水浴中1小时。每 10 分钟用 1 mL 移液管轻轻移液。</li>
	<li>向试管中加入 500 μL 5% FBS，并用 1 mL 移液管充分混合。</li>
	<li>将细胞悬液通过40μm无菌细胞过滤器过滤到新的1.5mL离心管中，并在4°C下以400× g 离心5分钟。</li>
	<li>小心地取出上清液，将细胞沉淀重悬于200μL解离试剂B中。</li>
	<li>置于37°C水浴中，再消化5分钟。在消化过程中每 2 分钟轻轻移液一次，以完全解离成单细胞悬浮液。</li>
	<li>使用200μL的5%FBS终止消化反应，并在4°C下以400× g 离心5分钟。</li>
	<li>弃去上清液，将细胞沉淀重悬于冰上的100μL1x PBS中。</li>
</ol>5. 细胞悬液检查和单细胞RNA测序
<ol><li>将 10 μL AO/PI 溶液加入 10 μL 单细胞悬浮液中，并用 20 μL 移液管轻轻混合。</li>
	<li>将 20 μL 混合物移入腔室载玻片中并等待 1 分钟。</li>
	<li>将载玻片装入自动细胞计数仪中。</li>
	<li>选择 AO/PI 活力 检测，然后等待质量报告。</li>
	<li>使用单细胞 3ʹ 试剂盒在单细胞控制器上的乳液中生成单细胞凝胶微球，并按照制造商的方案在热循环仪中扩增带条形码的 cDNA。 注：这里使用的是 Chromium Single Cell 3ʹReagent Kits v3。</li>
	<li>在具有双端 150 bp （PE150） 读数策略的测序仪上进行测序。使用软件应用程序 （Cell Ranger） 使用 mkfastq 管道将原始碱基调用文件转换为 fastq 文件。然后，使用 Cell Ranger 的 Count Pipeline 进行比对、过滤、条形码计数和唯一分子标识符 （UMI） 计数，以生成特征条形码矩阵13。</li>
	<li>使用 R 包 Seurat14 执行数据降维和可视化。<ol><li>首先，Seurat 的 Read10X（） 函数读取 Cell Ranger 管道的输出，然后使用计数矩阵创建 Seurat 对象。然后，通过修拉 的subset（） 函数过滤出表达<200或>4000个基因或线粒体基因比例超过10%的细胞。</li>
		<li>使用 NormalizeData （） 和 FindVariableFeatures（） 分别对数据进行归一化并识别 2000 个高度可变的特征。</li>
		<li>对缩放数据执行主成分分析 （PCA），并使用 dims = 30 和分辨率 = 0.5 对像元进行聚类。最后，使用 RunUMAP （） 函数可视化单细胞数据集，使用 FindAllMarkers（） 查找每个簇生物标志物。</li>
	</ol></li>
</ol>

小鼠颈动脉的两步细胞消化

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作者没有要披露的利益冲突。

在这里，我们提供了从野生型小鼠颈动脉制备高质量单细胞悬浮液的详细方案，其中构建了一种整合胶原酶/ DNase和胰蛋白酶消化过程的两步消化方法。经过对单细胞悬液的质量检查，我们发现它满足了单细胞测序的要求，细胞活力超过85%，细胞浓度高。此外，经过单细胞数据处理，成功检测到颈动脉中的多种细胞类型，包括VSMCs、成纤维细胞、ECs和Mφ/DCs。
目前制备颈动脉单?...

动脉粥样硬化是一种慢性炎症性疾病，与高血压、高脂血症和血流动力学等危险因素有关1。颈动脉分叉容易发生血流动力学改变，导致颈动脉斑块形成。颈动脉粥样硬化的临床表现可以是急性的，如卒中和短暂性脑缺血，也可以是慢性的，如复发性短暂性脑缺血和血管性痴呆2。从机械上讲，颈动脉斑块是病理条件下不同血管壁细胞与各种血细胞相互作用的结果。因此，揭示生理和病理条件下的颈动脉血管单细胞图谱对于预防和治疗颈动脉斑块的发展尤为重要。
单细胞 RNA 测序是生物学研究中最强大的技术之一，因为它具有超高分辨率和检测来自相同细胞类型生物体的细胞异质性 3,4。研究人员使用单细胞RNA测序在许多领域进行研究，例如心血管疾病5和癌症6。然而，快速准确地将组织分离成单细胞仍然是主要挑战之一。酶解离是一种常用的方法，主要包括胶原酶、木瓜蛋白酶、胰蛋白酶、DNase 和透明质酸酶。具体来说，胶原酶是单细胞消化的主要酶种，主要水解结缔组织中的胶原成分。不同的胶原酶类型适用于不同组织的解离，如乳腺7、肾小球8、虹膜角角9、膝关节10、主动脉11、肺12。由于不同组织独特的生理特性，使用相同的方法解离可能会在获取单个细胞时造成许多麻烦，例如细胞活力低、细胞数量少、细胞碎片大等。因此，针对不同组织的消化方法的发明对于制备高质量的单细胞悬浮液至关重要。
该方案旨在开发一种两步细胞消化方法，以制备野生型小鼠颈动脉的单细胞悬浮液。根据颈动脉的特点，我们将胶原酶/DNase与胰蛋白酶联合使用，以获得高质量的小鼠颈动脉单细胞悬液，因为胶原酶可以水解颈动脉组织的胶原蛋白，颈动脉组织被胰蛋白酶进一步消化成单细胞悬浮液。采用吖啶橙/碘化丙啶（AO/PI）双荧光计数检测细胞活力和浓度，发现单细胞悬液满足单细胞测序要求，细胞活力超过85%，细胞浓度高。经过单细胞数据处理，在消化后的正常小鼠颈动脉中鉴定出血管平滑肌细胞（VSMCs）、成纤维细胞、内皮细胞（ECs）和巨噬细胞和树突状细胞（Mφ/DCs）等4种细胞类型。该协议的优点是：1）可以识别颈动脉中的多种细胞类型，2）细胞活力保存良好，3）无需特殊设备即可轻松重复。该协议适用于有兴趣研究小鼠颈动脉单细胞多组学的研究人员。该方案还可能有助于通过适当的修改解离其他血管。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% EDTA-free trypsin</td>
			<td>Beyotime</td>
			<td>C0205</td>
			<td>Dilute 1 mL of 0.25% EDTA-free trypsin into 1 mL of 1x PBS.</td>
		</tr>
		<tr>
			<td>0.9% NaCl saline solution</td>
			<td>Beyotime</td>
			<td>ST341</td>
			<td>Dilute the heparin sodium solution into a final concentration of 10 mg/mL</td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>&#160;SKJYLEAN</td>
			<td>sk-r009</td>
			<td>To perform cardiac perfusion</td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>KIRGEN</td>
			<td>KG2211W</td>
			<td>To centrifuge the tissue piece and cell suspension</td>
		</tr>
		<tr>
			<td>20 mL syringes</td>
			<td>SKJYLEAN</td>
			<td>sk-r013</td>
			<td>To perform cardiac perfusion</td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>JETBIOFIL</td>
			<td>css010040</td>
			<td>To filter undigested tissue fragments</td>
		</tr>
		<tr>
			<td>AO/PI kit</td>
			<td>Hengrui Biological</td>
			<td>RE010212</td>
			<td>To identify whether the cell is alive or dead</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>Mira FL</td>
			<td>To analyze the cell morphology and cell viability of digested carotid vascular cells</td>
		</tr>
		<tr>
			<td>Cell Ranger software</td>
			<td>10&#215; Genomics</td>
			<td>3.0.2</td>
			<td>To process Chromium single-cell RNA-seq output and perform clustering and gene expression analysis</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39;Reagent Kits v3</td>
			<td>10&#215; Genomics</td>
			<td>1000075</td>
			<td>To prepare single-cell RNA-seq libraries of single-cell suspension</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td>Dilute with HBSS to a final concentration of 125 CDU/mL</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington</td>
			<td>LS002140</td>
			<td>Dilute with HBSS to a final concentration of 60 U/mL</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>HyClone</td>
			<td>SH30088.03</td>
			<td>Termination of the digestion reaction</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution&#160;</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>Store at the room temperture</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Solarbio Life Science</td>
			<td>H8060</td>
			<td>Dilute with 0.9% NaCl to a final concentration of 10 mg/mL</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002560</td>
			<td>Applied for spining down the tissues and cell pellets</td>
		</tr>
		<tr>
			<td>NovaSeq 6000&#160;</td>
			<td>Illumina</td>
			<td>N/A</td>
			<td>Sequencer</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Solarbio Life Science</td>
			<td>P1000</td>
			<td>Used for cardiac perfusion and resuspension of cells</td>
		</tr>
		<tr>
			<td>Seurat package- R</td>
			<td>Satija Lab</td>
			<td>3.1.2</td>
			<td>To performed dimensionality reduction, visualization, and analysis of scRNA-sequencing data</td>
		</tr>
		<tr>
			<td>Six-well cell culture plates</td>
			<td>NEST</td>
			<td>703002</td>
			<td>Place the vascular tissue</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Jinghong</td>
			<td>DK-S22</td>
			<td>Keep the digestion temperature at 37 &#176;C</td>
		</tr>
	</tbody>
</table>

preparation of a single-cell suspension from mouse carotid arteries for single-cell sequencing

颈动脉是颈部的主要血管，为大脑提供血液和氧气，但当颈动脉被斑块堵塞时，就会发生颈动脉狭窄。在单细胞水平上揭示颈动脉的细胞组成对于治疗颈动脉粥样硬化至关重要。然而，目前尚无现成的方案用于制备颈动脉单细胞悬浮液。为了获得在单细胞水平上解离正常颈动脉且对细胞损伤较小的合适方案，我们设计了一种两步消化方法，通过整合胶原酶/DNase 和胰蛋白酶的消化过程。采用吖啶橙/碘化丙啶（AO/PI）双荧光计数检测细胞活力和浓度，发现单细胞悬液满足单细胞测序要求，细胞活力超过85%，细胞浓度高。单细胞数据处理后，在每个颈动脉细胞中检测到每个细胞的中位数~2500个转录本。值得注意的是，可同时检测到正常颈动脉的多种细胞类型，包括血管平滑肌细胞 （VSMC）、成纤维细胞、内皮细胞 （EC） 以及巨噬细胞和树突状细胞 （Mφ/DC）。该方案可用于制备具有适当修饰的来自其他组织的血管的单细胞悬浮液。

Atherosclerosis is a chronic inflammatory disease associated with risk factors such as high blood pressure, hyperlipidemia, and hemodynamics1. Carotid artery bifurcations are prone to hemodynamic changes and lead to carotid plaque formation. The clinical presentation of carotid atherosclerosis can be acute such as stroke and transient cerebral ischemia, or chronic such as recurrent transient cerebral ischemia and vascular dementia2. Mechanically, carotid plaque is the outcome of the interaction between different vessel wall cells and various blood cells under pathological conditions. Therefore, revealing the single-cell atlas of carotid vessels under physiological and pathological conditions is particularly important for preventing and treating carotid plaque development.
Single-cell RNA sequencing is one of the most powerful technologies of biological research because of its ultrahigh resolution and detection of cell heterogeneity from the same cell type of organisms3,4. Researchers have used single-cell RNA sequencing to conduct research in many fields, such as cardiovascular disease5 and cancer6. However, quickly and accurately separating tissues into single cells is still one of the main challenges. Enzymatic dissociation is a commonly used method that dominantly includes collagenase, papain, trypsin, DNase, and hyaluronidase. Specifically, collagenases are the major enzyme species for single-cell digestion, mainly hydrolyzing collagen components in connective tissues. Different collagenase types are applicable for the dissociation of different tissues, such as the mammary gland7, glomerulus8, iridocorneal angle9, knee joint10, aorta11, and lung12. Due to the unique physiological properties of different tissues, dissociation using the same method may cause many troubles in acquiring single cells, such as low cell viability, low cell number, and large cell debris. Therefore, the invention of digestion methods for different tissues is essential for preparing high-quality single-cell suspensions.
This protocol aims to develop a two-step cell digestion method to prepare a single-cell suspension of the carotid artery of wild-type mice. According to the characteristics of the carotid artery, we combined collagenase/DNase with trypsin to obtain a high-quality single-cell suspension of the mouse carotid artery because collagenase can hydrolyze collagen of the carotid tissues, which was further digested by trypsin into a single-cell suspension. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, four cell types, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were identified in normal mouse carotid arteries after digestion. The advantages of this protocol are that: 1) multiple cell types in the carotid artery can be identified, 2) cell viability is well preserved, and 3) it can be easily repeated without special equipment. This protocol is suitable for researchers who are interested in studying single-cell multiomics of the mouse carotid arteries. This protocol may also be helpful in the dissociation of other blood vessels with appropriate modifications.

作者聚焦：血管组织解离和探索用于靶向治疗的单细胞亚簇 

从小鼠颈动脉制备用于单细胞测序的单细胞悬液

Low solubility and the larger cell debrief are the main difficulties of vascular tissue dissociation. Our two steps or digestion method is very suitable for carotid tissue dissociation and may be used to prepare single cell suspicions for other vessels with minor modifications, making it very helpful in cardiovascular research. This protocol is suitable for obtaining multiple cell types in the carotid artery, and it can be easily repeated without special equipment.In the future, we aim to use cell subclasses discovered by single cell ion sequencing for targeted therapy of cardiovascular disease.

该方案描述了一种用于制备小鼠颈动脉单细胞悬浮液的两步细胞消化方法（图1）。这种两步细胞消化方法将胶原酶/DNase与胰蛋白酶相结合，可有效解离小鼠颈动脉血管壁，获得用于单细胞测序的高质量单细胞悬液。解离后，通过自动细胞计数仪计算细胞浓度和细胞活力。明场显示消化后颈动脉血管细胞的形态，AOPI双荧光染色同时检测细胞活力（图2A，B</strong...

Watch this Scientific Journal Video about Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy at JoVE.com

在这里，我们描述了一种两步细胞消化方案，用于制备小鼠颈动脉的单细胞悬浮液。

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

溶解度低和细胞分布较大是血管组织解离的主要困难。我们的两步或消化方法非常适合颈动脉组织解离，可用于为其他血管准备单细胞怀疑，只需稍作修改，对心血管研究非常有帮助。该方案适用于在颈动脉中获得多种细胞类型，无需特殊设备即可轻松重复。未来，我们的目标是利用单细胞离子测序发现的细胞亚类进行心血管疾病的靶向治疗。

preparation-single-cell-suspension-from-mouse-carotid-arteries-for

Research

JoVE Journal

Biology

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

2.0K 次观看.  Soochow University. 溶解度低和细胞分布较大是血管组织解离的主要困难。我们的两步或消化方法非常适合颈动脉组织解离，可用于为其他血管准备单细胞怀疑，只需稍作修改，对心血管研究非常有帮助。该方案适用于在颈动脉中获得多种细胞类型，无需特殊设备即可轻松重复。未来，我们的目标是利用单细胞离子测序发现的细胞亚类进行心血管疾病的靶向治疗。