Name: isolation and culture of primary aortic endothelial cells from miniature pigs
Uploaded: 2019-08-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs at JoVE.com

这项工作得到了广东省自然科学基金的资助,资助/奖励编号:2016A030313028;广东省医学科学研究基金会,资助/奖励编号:B2018003;深圳市科技基金会,授予/奖励编号:JCYJ20180306172449376, JCYJ20180306172459580, JCJY2016022292204849975, GJHZ2017031417171357556, JCYJ20160103000011 和 JCYJ2016016042822040404444;深圳市龙华区科技基金会,授奖/奖励编号:2017013;国家重点研发项目,授权/奖励编号:2017YFC1103704;深圳三明医药项目,授予/奖励编号:SZSM201412020;广东省高水平医院建设专项资金(2019年);深圳市高水平医学学科建设基金,助学金/奖励号:2016031638;深圳市卫生和计划生育委员会,授予/奖励编号:SZXJ2017021和SZXJ2018059。我们感谢深圳大学的张汉成和赵志成协助编写手稿。

动物的使用是深圳市第二人民医院伦理审查委员会根据动物福利原则批准的。
1. 动物、介质和缓冲液的准备
<ol><li>准备微型猪。 注:所有微型猪都是中国五指山或巴马猪(雄性)。猪的年龄和体重分别为100天~8天和5.7公斤=1.0公斤(均值=SD,n = 3)。</li>
	<li>准备培养基:内皮细胞培养基(ECM)辅以10%(v/v)热灭活胎儿牛血清(FBS),1%(v/v)青霉素/链霉素(P/S)和1%(v/v)内皮细胞生长补充剂(ECGS)。</l...

<ol>
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内皮细胞通常用于血管功能障碍、糖尿病、组织再生、移植和癌症14、15、16、17、18的研究。为了了解和描述EC在这些疾病中的生物学和功能,已经报道了许多方法分离不同患病器官或组织的EC8,19,20<...

移植器官短缺是一个突出的世界性问题。根据中国红十字会的数据,2018年,中国只有少数期器官衰竭患者获得了合适的器官。
异体移植是解决器官短缺问题的一种有希望的途径。猪器官被认为是最适合人类的器官,因为解剖学和生理上的相似性2,3。猪异种移植的失败很大程度上与灵长类动物的免疫排斥和凝固反应有关。猪内皮细胞(ECs)至关重要,因为这些细胞是第一个与灵长类免疫系统相互作用的细胞,包括抗体、补体、细胞因子和免疫细胞(例如T细胞、B细胞和巨噬细胞)4、5。猪EC在猪器官和小岛异种移植中起着至关重要的作用。因此,ECs是研究猪移植物的排斥和凝固反应的重要细胞。异种移植研究需要分离高质量的猪E。
在试图从不同的器官(例如心脏、肾脏、肝脏和大头塔)中分离EC时,在异种移植设置8、9、10、11、12中报告了几种方案。然而,保持隔离的EC的超纯文化是标准协议中的一个突出问题。在目前的研究中,消化溶液浓度增加、消化时间和刮削强度不当,可能导致成纤维细胞污染增加。此外,对微型猪的分离方法的研究较少。在这里,我们描述了一种优化的酶法,从微型猪(武志山或巴马)中分离出高度纯化的PAE。该协议中的几个步骤已经设计用于减少成纤维细胞污染和获得高纯度的ECs。

<table border="0" cellpadding="0" cellspacing="0" style="width:1163px;" width="1164">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">BD FACSAria II</td>
			<td style="width:424px;">BD Bioscience</td>
			<td style="width:142px;"></td>
			<td style="width:318px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Boneforceps</td>
			<td style="width:424px;">Beijing HeLi KeChuang Technology Development CO.,Ltd. China</td>
			<td style="width:142px;">HL-YGQ&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">BOON Disposable Syringe (10ml)</td>
			<td style="width:424px;">Jiangsu Yile medical Article Co., Ltd. China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">CD31-FITC antibody</td>
			<td style="width:424px;">Bio-Rad</td>
			<td style="width:142px;">MCA1746F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Cell scraper</td>
			<td style="width:424px;">Corning&#160;</td>
			<td style="width:142px;">3010#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Collagenase IV</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:142px;">C5138#-1G</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:280px;">Compound ketamine injection&#160;</td>
			<td style="width:424px;">Veterinary Pharmaceutical Factory of Shenyang, China</td>
			<td style="width:142px;"></td>
			<td style="width:318px;">Ketamine Hydrochloride&#65306;0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride&#65306;1mg/2ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:424px;">Life Technologies</td>
			<td style="width:142px;">D1306#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">DMEM</td>
			<td style="width:424px;">Life Technologies</td>
			<td style="width:142px;">11965118#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">ECM</td>
			<td style="width:424px;">Sciencell</td>
			<td style="width:142px;">1001#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">ECGS</td>
			<td style="width:424px;">Sciencell</td>
			<td style="width:142px;">1052#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Eppendorf Snap-Cap Microtube(1.5mL)&#160;</td>
			<td style="width:424px;">AXYGEN</td>
			<td style="width:142px;">MCT-150-C#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Falcon 100mm Cell Culture Dish</td>
			<td style="width:424px;">Corning</td>
			<td style="width:142px;">353003#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Fetal Bovine Serum</td>
			<td style="width:424px;">GIBCO</td>
			<td style="width:142px;">10270-106#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Flowjo v10.0</td>
			<td style="width:424px;"></td>
			<td style="width:142px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Forceps&#160;</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Heparin sodium</td>
			<td style="width:424px;">Jiangsu WanBang biopharmaceutical Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Iodine tincture</td>
			<td style="width:424px;">Guilin LiFeng Medical Supplies Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Miniature Pig (Bama or Wuzhishan)</td>
			<td style="width:424px;">Kang Yi Ecological Agriculture Co., Ltd, China</td>
			<td style="width:142px;"></td>
			<td style="width:318px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Mshot microscope&#160;</td>
			<td style="width:424px;">Guangzhou Micro-shot Technology Co., Ltd.</td>
			<td style="width:142px;">M152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Petri Dishes (150 x 15 mm)</td>
			<td style="width:424px;">Biologixgroup</td>
			<td style="width:142px;">66-1515#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Penicillin/Streptomycin</td>
			<td style="width:424px;">Life Technologies</td>
			<td style="width:142px;">15070063#</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:280px;">Rectangular Canted Neck Cell Culture Flask with Vent Cap &#65288;T25&#65289;</td>
			<td style="width:424px;">Corning&#160;</td>
			<td style="width:142px;">3289#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Scissors</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Serological Transfer Pipettes (10ml)</td>
			<td style="width:424px;">JET Biofil</td>
			<td style="width:142px;">GSP010010#&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Pasteur Pipette</td>
			<td style="width:424px;">GeneBrick</td>
			<td style="width:142px;">GY0025#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Syringe Filter (0.22&#181;m)</td>
			<td style="width:424px;">Millipore</td>
			<td style="width:142px;">SLGV033RS#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Surgical scalpel</td>
			<td style="width:424px;">ShangHai medical instruments Co.,Ltd.China</td>
			<td style="width:142px;">22#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Surgical suture</td>
			<td style="width:424px;">Shanghai Pudong Jinhuan Medical Supplies Co., Ltd</td>
			<td style="width:142px;">5-0#</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Syringe&#65288;5mL&#65289;</td>
			<td style="width:424px;">Shengguang Medical Instrument Co., Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:424px;">GIBCO</td>
			<td style="width:142px;">25200056#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">75% Medical alcohol</td>
			<td style="width:424px;">Guilin LiFeng Medical Supplies Co.,Ltd.China</td>
			<td style="width:142px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">20 x PBS solution (pH 7.4,Nuclease free)</td>
			<td style="width:424px;">Sangon Biotech</td>
			<td style="width:142px;">B540627#</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Medical disinfectant 84 liquid</td>
			<td style="width:424px;">Sichuan Province Yijieshi Medical Technology Co., Ltd</td>
			<td style="width:142px;">450ml/bottle</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary aortic endothelial cells from miniature pigs

异种移植是解决终末期器官衰竭患者人体器官短缺的一种有希望的方法,猪被认为是合适的器官来源。免疫排斥和凝固是异种移植成功的两个主要障碍。血管内皮细胞(EC)损伤和功能障碍对异种移植中炎症和凝血反应的发展具有重要意义。因此,分离猪主动脉内皮细胞(pAECs)对于研究免疫排斥和凝固反应是必要的。在这里,我们开发了一种简单的酶法,用于从微型猪中分离、表征和扩展高度纯化的PAE。首先,微型猪被用氯胺酮麻醉,并切除了一条大头塔的长度。其次,主肠的内皮表面暴露于0.005%胶原酶IV消化溶液15分钟。 第三,主肠的内皮表面只刮一个方向与细胞刮刀(<10次),并没有压缩过程中刮。最后,通过流式细胞测定与抗CD31抗体测定第3天和通道1和通道2之后的分离的PAEC。CD31阳性细胞的百分比分别为97.4%~1.2%、94.4%~1.1%和92.4%~1.7%(均值=SD)。胶原酶IV的浓度、消化时间、方向、刮擦的频率和强度对于减少成纤维细胞污染、获得高纯度和大量EC至关重要。总之,我们的酶法是一种从微型猪主主塔中分离ECs的高效方法,细胞可以在体外扩展,以研究异种移植中的免疫和凝固反应。

我们的方法旨在提供一种有效的方法,从主头塔中分离出高度纯化的E从微型猪。主数手术的过程如图1所示。第一步是从猪中切除了整个主塔。防止其他细胞或细菌污染是非常重要的。因此,不要伤害其他器官或组织,以防非靶向细胞或细菌污染主方,用预冷却洗涤缓冲液3x清洗主塔(图1B-D)。保持细胞活力的另一个关键步骤是尽量减少从获得手术...

Watch this Scientific Journal Video about Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs at JoVE.com

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

介绍了一种从微型猪中分离原发猪主动脉内皮细胞(pAECs)的有效酶法。分离的初级pAEC可用于研究异种移植中的免疫和凝固反应。

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

该方法是将内皮细胞与微型猪隔离的高效方法。细胞也可以扩大，研究异种移植中的免疫和凝固反应。技术的主要优点是，从猪体内分离出高度纯化的内皮细胞不仅简单、高效，而且在通过时更便于保存其他pAEC。在确认麻醉猪对疼痛反射缺乏反应后，用75%的医用酒精和一剂碘洗猪腹壁三次。最后一次洗涤后，用手术刀做腹部切口，露出劣质的维纳卡瓦，并使用五毫升注射器将5000单位肝素钠注射到劣质的维纳卡瓦中。五分钟后，将导管插入腹部主动脉，并使用手术刀将隔膜插入。在第二名调查员的帮助下，切除左心室，用骨钳切除肋骨，露出心脏和肺部。找到心肺后心大母，夹住末端。用剪刀切除大干，用预冷洗涤缓冲液洗一次。用无菌钳和剪刀去除大干周围的任何多余的组织，将容器放入含有新鲜预冷洗涤缓冲液的50毫升离心管中，转移到实验室。对于猪大动脉内皮细胞分离，在无菌条件下将猪大动脉转移到150毫米的培养皿中，然后轻轻取出血管的两端。用无菌钳子和剪刀修剪容器被夹住的任何额外组织，并使用 20 至 50 毫升的新鲜洗涤缓冲液冲洗大干的内外。接下来，通过手术缝合通过大血管，并松散地绑住容器的一端，保持缝合线在流明内。用钳子轻轻固定靠近绑端的大动脉，然后缓慢地拉下手术缝合线，反转大动脉，直到大动脉的内皮表面暴露。每次洗涤用 10 毫升新鲜洗涤缓冲液洗大动脉的内皮表面三次，将大动脉放入 15 毫升离心管中。用10毫升37摄氏度加热0.005%胶原酶IV消化液，在37摄氏度下孵育组织15分钟。在孵育结束时，将整个管内物转移到100毫米培养盘中，用10至15毫升预冷却停止缓冲液停止消化。使用细胞刮刀，轻轻地从容器内表面取出大动脉内皮细胞，每次洗涤用五毫升洗涤缓冲液洗涤容器三次。上次洗涤后，将上清液转移到50毫升离心管中，用5毫升的新鲜洗涤缓冲液洗涤培养皿底部两次，每次洗涤，将洗涤液拉入50毫升管中。通过离心收集细胞，并丢弃所有，但最后10毫升的上经剂。加入20毫升的洗涤缓冲液以重新填充颗粒，注意避免气泡，并收集细胞与另一个离心。将颗粒重新在一毫升细胞培养中进行计数，并在每25厘米平方的烧瓶浓度中，以1倍10至6个细胞的镀块。然后将细胞培养孵化器中的细胞在5%的二氧化碳中放在37摄氏度，每两到三天刷新一次培养基。隔离后，在第1天、第1天和2天检查内皮细胞，并在第1天和2日通过后检查污染及其形态评估。使用抗CD31-FITC抗体进行流动细胞计量分析表明，在培养的第三天，一般大于97%的内皮细胞标记阳性群体，即使在两次通过后，其纯度也保持在90%以上。大动脉的内皮表面只刮向一个方向，用细胞刮刀刮，不到10次，在刮擦过程中没有压缩。最好的答案，优化的pAECs分离方法，我们可以使用纯化的pAECs进行个别实验，并研究异种移植中的免疫排斥和凝固反应。

Research

JoVE Journal

Biology

Isolation of Valvular Endothelial Cells

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

科研

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...