Name: characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood
Uploaded: 2022-01-06T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

作者希望感谢NIH / NHLBI R00 HL129068的资助。

所有动物研究均由威斯康星医学院和妙佑医疗国际各自的机构动物护理和使用委员会 （IACUC） 批准。
注意：在这项研究中，使用了约克郡/兰德雷斯/杜洛克杂交家猪（Sus homeus），雄性和雌性，40-80公斤，3-6个月大。
1.猪外周血采集
<ol><li>准备材料。<ol><li>在无菌盐水中将肝素溶液稀释至100U / mL。</li>
		<li>向两个 50 mL 锥形管和两个 60 mL...

<ol>
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BOEC是一种强大的工具，可用于各种科学和治疗方法7，8，16。BOEC已被用于分析EC基因表达，以阐明导致血管疾病和癌症发展的关键因素5，19，20，21。BOEC还被用于治疗应用，如血管再生和基因传递22，<sup cl...

内皮是一种高度复杂的动态结构，是血管壁的重要组成部分。它排列在血管的内表面，在循环血液和周围组织之间提供物理界面。已知这种异质结构执行各种功能，例如血管生成，炎症，血管调节和止血1，2，3，4。人脐静脉内皮细胞是一种被广泛研究的细胞类型，用于评估内皮细胞的功能。然而，患者特异性批次变异性、表型不一致和最小分裂效率表明需要确定可以改善所有这些特征的细胞来源5。
获得原代内皮细胞的均质群体在技术上可能具有挑战性，并且原代内皮细胞不具有高增殖能力6。因此，为了研究血管再生和评估病理生理过程，各个小组试图获得和评估来自外周血的不同类型的内皮细胞，例如内皮祖细胞（EPC）或血液生长内皮细胞（BOEC）6，7，8，9.纺锤形的早期EPC起源于造血干细胞（HSC），具有有限的生长效力和有限的血管生成能力，无法产生成熟的内皮细胞。此外，它们与炎性单核细胞非常相似。此外，它们进一步分化成功能性、增殖、成熟的内皮细胞的能力仍有争议6，7，9，10。外周血单核细胞 （PBMC） 的连续培养可产生称为晚期生长 EPC、BOEC 或内皮集落形成细胞 （ECFC） 的二次细胞群6，7，9，10。Medina等人在2018年承认EPC的局限性，其命名的模糊性，以及与EPC11下连续分组的许多不同细胞类型普遍缺乏一致性。相比之下，BOEC因其在血管修复，健康和疾病以及细胞治疗中的作用而得到认可。这些细胞的进一步研究和治疗使用将依赖于方案，以一致地从循环祖细胞中获取这些细胞类型。
BOEC等原代细胞可用作替代细胞，以获得高度增殖的成熟内皮细胞6。BOEC在表型上与早期EPC不同，并表现出典型的内皮特征，例如鹅卵石形态以及粘附连接和洞穴的表达12。Hebbel等人的基因分析13，14，15发现BOEC或ECFC是真正的内皮细胞，因为它们促进微血管和大血管的形成。因此，BOEC可以用作评估病理生理过程和遗传变异的工具16。它们也被认为是血管再生细胞疗法的极好细胞来源17。因此，一个标准化的方案来持续获得这些高度增殖的细胞是必不可少的。
虽然BOEC为研究人类病理生理和遗传变异提供了强大的工具，但更同质的BOEC来源可能会提供更强大和可靠的实验和治疗结果。通过使用来自在相似条件下饲养的遗传相似动物的异种细胞源，可以实现卓越的同质性18。虽然异种细胞来源容易引发宿主免疫反应，但正在开发免疫调节策略，目的是产生免疫相容的动物和动物产品，包括细胞。特别是猪，是外周血的丰富来源，由于与人类的解剖和生理相似性，通常用于研究医疗设备和其他疗法。因此，本研究完善了从猪外周血中分离和扩增高度增殖的BOEC的方案。下面详述的方案是一种从相对少量的血液中获取大量 BOEC 的简单可靠的方法。培养物可以通过多次传代扩增，以从单个血液样本中产生数百万个细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>19 G needle</td>
			<td>Covidien</td>
			<td>1188818112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringes</td>
			<td>Covidien</td>
			<td>8881560125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride solution (0.8%)</td>
			<td>Stemcell Technologies</td>
			<td>07850</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-253-839</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 culture medium</td>
			<td>Lonza Walkersville</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Extension tube</td>
			<td>Hanna Pharmaceutical Supply Co.</td>
			<td>03382C6227</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlas Biologicals</td>
			<td>F-0500-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque 1077</td>
			<td>Cytiva</td>
			<td>17144003</td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>Heparin sodium injection (1,000 units/mL)</td>
			<td>Pfizer</td>
			<td>00069-0058-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Gibco</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Eppendorf</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Gibco</td>
			<td>15230-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin pipette</td>
			<td>Celltreat Scientific</td>
			<td>229280</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood

内皮是一种动态的综合结构，在血管生成、止血、炎症和体内平衡等许多生理功能中起重要作用。内皮在动脉粥样硬化、高血压和糖尿病等病理生理学中也起着重要作用。内皮细胞形成血液和淋巴管的内层，在结构和功能上表现出异质性。各种小组已经评估了来源于人外周血的内皮细胞的功能，重点是来源于造血干细胞或成熟血液生长内皮细胞（或内皮集落形成细胞）的内皮祖细胞。这些细胞为治疗和疾病建模提供了自体资源。异种细胞可以提供另一种治疗来源，因为它们的可用性和同质性通过使用在类似条件下饲养的遗传相似的动物来实现。因此，已经提出了一种从猪外周血中分离和扩增高度增殖的血液生长内皮细胞的稳健方案。这些细胞可用于多种应用，如心血管组织工程、细胞治疗、疾病建模、药物筛选、研究内皮细胞生物学以及 体外 共培养，以研究异种移植中的炎症和凝血反应。

从培养开始到观察到BOEC菌落，观察培养细胞的形态（图1）。较小的贴壁细胞群开始附着在培养皿上并生长，而非贴壁细胞随着培养基的变化而被移除（图1B）。菌落在第6天首次出现，作为从中心点向外放射状增殖的内皮样细胞的集合（图1D）。随着培养的进行，细胞集落变得更加致密，并显示出类似于成熟内皮细胞的鹅卵石形态...

Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

猪外周血血液生长内皮细胞（BOEC）的表征

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

该方案提供高度增殖的血液生长内皮细胞，可用于各种组织工程、细胞治疗和疾病建模研究。这种技术始终如一地从一次抽血中产生许多细胞。为了使该技术取得成功，尽快将细胞快速放入培养基和培养箱环境中至关重要。首先，在无菌盐水中将肝素溶液稀释至每毫升100单位。向两个 50 毫升锥形管和两个 60 毫升注射器中各添加三至四毫升肝素溶液。然后，将未稀释的每毫升肝素溶液1， 000单位吸入延长管中。将 19 号针头连接到充满肝素的延长管的一端，将含肝素的 60 毫升注射器连接到延长管的另一端。接下来，用Betadine磨砂溶液清洁麻醉猪的股骨腹股沟区域。用 19 号针刺穿股静脉或动脉，以每秒 1 到 2 毫升的速度，缓慢地将 50 毫升血液吸入注射器。将针头留在原位，立即扭结延长管并断开 60 毫升注射器。将血液缓慢转移到含肝素的50毫升锥形管中。盖紧锥形管，轻轻倒置管几次混合，然后将其放在冰上。将剩余的含肝素的60毫升注射器连接到延长管。解开延长管，慢慢地将额外的50毫升血液吸入注射器中。立即从动脉或静脉中取出针头，并将延长管中的剩余血液缓慢抽入注射器。断开60毫升注射器与延长管的连接，然后将血液缓慢转移到剩余的含肝素的50毫升锥形管中。盖紧50毫升锥形管，轻轻倒置几次混合，然后放在冰上。对猪的股骨腹股沟区域施加压力止血。用PBS在四个50毫升的锥形管中一对一稀释血液。将 25 毫升室温即用型密度梯度溶液加入八个 50 毫升锥形管中。通过将管与移液器尖端成锐角，在每个含有密度梯度溶液的50毫升锥形管内缓慢移取25毫升血盐水溶液。将试管在560g和室温下离心30分钟。用细移液管从每个管中小心地收集血沉棕黄层，这是密度梯度溶液上方和透明血浆下方的单核细胞浑浊层，并将其均匀地分布到两个新的 50 毫升锥形管中。丢弃含有管的密度梯度溶液。要用纤连蛋白涂覆六孔板，通过在无菌水中将其稀释至每毫升一毫克来制备人血浆纤连蛋白储备溶液。通过在三毫升PBS中稀释600微升纤连蛋白储备溶液，制成3.6毫升纤连蛋白的工作溶液。将600微升纤连蛋白工作溶液转移到六孔板的每个孔中，轻轻摇动直至均匀涂覆。并轻轻地从每个孔中吸出溶液。要在等待纤连蛋白包被的同时清洗单核细胞，请在试管中加入多达 45 毫升的 PBS，在 560 X g 和 4 摄氏度下以低断裂离心五分钟。从两个管中吸出上清液。将每个细胞沉淀重悬于25毫升PBS中。将每个细胞沉淀重悬于5毫升PBS中，并向每个试管中加入15毫升0.8%氯化铵溶液。像以前一样通过添加PBS清洗细胞。从两个管中吸出上清液。将每个细胞沉淀重悬于25毫升PBS中。将每个细胞沉淀重悬于补充有10%FBS和1X抗生素 - 抗真菌溶液的6毫升EGM-2培养基中。轻轻地将两毫升细胞悬液转移到纤连蛋白包被的六孔板的每个孔中，并轻轻摇动直至均匀包被。接种细胞24小时后，轻轻向每个孔中加入一毫升新鲜培养基。第二天，从每个孔中轻轻吸出1.5毫升培养基，并用1.5毫升新鲜培养基代替。在接下来的五天里，用每孔两毫升新鲜培养基轻轻更换培养基。在低功率4X物镜显微镜下定期观察细胞培养物。从培养开始到观察到BOEC菌落，观察培养细胞的形态。较小的贴壁细胞群开始附着在培养皿上并生长，而非贴壁细胞则随着培养基的变化而被移除。菌落在第六天首次出现，作为从中心点向外放射状增殖的内皮样细胞的集合。随着培养的进行，细胞集落变得更加致密，并显示出类似于成熟内皮细胞的鹅卵石形态。二维相衬显微镜用于对无血清培养基和完全生长培养基中的管形成进行成像。两种培养基条件下的细胞都经历了形态分化，并迅速组织成广泛的毛细管状结构网络。BOECs还通过表达成熟的内皮细胞标志物CD31或血小板内皮细胞粘附分子之一来表征。BOEC表现出CD31的均匀表达。此外，与阳性对照组相比，具有CD14 AF 700抗体的BOEC的流式细胞术分析证实了EPC的缺失，因为与PBMC的阳性对照组相比，单核细胞标志物CD14染色呈阴性。 CD14染色阳性显示为蓝色，未染色细胞显示为红色。在采血过程中，必须收集尽可能多的血沉棕黄层，同时尽可能少地收集相邻层。该技术使研究人员能够研究使用具有自体血液生长内皮细胞的猪模型内皮化植入式心血管装置的新策略。

Research

JoVE Journal

Bioengineering

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

科研

生物工程

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Blood Flow Imaging with Ultrafast Doppler

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...