从肺源性样品中分离和纯化微血管和大血管内皮细胞
1Department of Medical, Oral and Biotechnological Sciences,“G. d’Annunzio” University of Chieti-Pescara, 2Center on Advanced Studies and Technology (CAST),“G. d’Annunzio” University of Chieti-Pescara, 3Department of Medicine and Aging Sciences,University “G. d’Annunzio” of Chieti-Pescara, 4U.O. di Chirurgia Toracica e dei Trapianti di Polmone,Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico di Milano, 5Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti,Università degli Studi di Milano, 6Department of Pharmaceutical Sciences,Università degli Studi di Milano, 7Unit of Cell and Molecular Biology in Cardiovascular Diseases,Centro Cardiologico Monzino IRCCS
Summary
尽管具有挑战性，但肺内皮细胞的分离对于肺部炎症的研究至关重要。本协议描述了大血管和微血管内皮细胞的高产量，高纯度分离的程序。
Abstract
从健康和患病组织和器官中分离的细胞的可用性是个性化医疗方法的关键要素。尽管生物样本库可以为生物医学研究提供广泛的原代细胞和永生化细胞，但这些并不能涵盖所有实验需求，特别是与特定疾病或基因型相关的实验需求。血管内皮细胞（ECs）是免疫炎症反应的关键组成部分，因此在多种疾病的发病机制中起着核心作用。值得注意的是，来自不同位点的EC表现出不同的生化和功能特性，使得特定EC类型（即大血管，微血管，动脉和静脉）的可用性对于设计可靠的实验至关重要。在这里，详细说明了从肺动脉和肺实质中获得高产量、几乎纯的人大血管和微血管内皮细胞的简单程序。任何实验室都可以以相对较低的成本轻松复制该方法，以实现独立于商业来源并获得尚不可用的EC表型/基因型。
Introduction
血管内皮排列在血管的内表面。它在调节血液凝固、血管张力和免疫炎症反应中起关键作用1，2，3，4。尽管从人类标本中分离的内皮细胞（ECs）的培养对于研究目的至关重要，但必须指出的是，来自不同血管（动脉，静脉，毛细血管）的EC具有特定的功能。这些不能完全由人脐静脉内皮细胞（HUVEC）概括，后者很容易获得并广泛用于血管内皮病理生理学研究5，6。例如，人肺微血管内皮细胞（HLMVECs）通过控制白细胞募集和积累在肺部炎症中起关键作用4，7。因此，旨在以高保真度再现肺部炎症的实验环境应包括HLMVEC。另一方面，可以在几种病理中观察到EC功能障碍;因此，来自患者的EC对于建立可靠的疾病体外模型至关重要。例如，从囊性纤维化（CF）患者的肺中解剖出的肺动脉（HPAEC）中分离EC碎片，使我们能够发现这种疾病内皮功能障碍的机制8，9。
因此，旨在优化疾病状态下不同来源/器官的EC分离的方案对于为研究人员提供有价值的研究工具至关重要，特别是当这些工具无法商业化时。HLMVEC和HPAEC分离协议先前已报道10，11，12，13，14，15，16，17，18，19。在所有情况下，肺标本的酶消化导致混合细胞群，使用特设选择性培养基和基于磁珠或细胞术的细胞分选进行纯化。这些方案的进一步优化必须解决EC分离中的两个主要问题：（1）细胞和组织污染，应尽早解决培养通道，以尽量减少EC复制衰老20;（2）初级EC分离株的产量低。
本研究描述了一种用于HLMVEC和HPAEC的高产量、高纯度分离的新方案。该程序很容易应用，只需几个步骤即可获得几乎纯的大血管和微血管 EC。
Protocol
这项研究获得批准，协议遵循基耶蒂-佩斯卡拉大学人类研究伦理委员会（#237_2018bis）的指导方针。 图 1说明了从肺实质或肺动脉的节段（1-3cm长）中分离出内皮细胞，这些细胞来自因各种原因（例如气胸或肺叶切除术）接受胸外科手术的去识别人类受试者（书面同意）。在后一种情况下，外科医生还收集了肺动脉段。值得注意的是，外科医生被准确地指示收集无癌症样本。?…
Representative Results
HLMEC 隔离HLMVEC分离过程中的主要问题是污染细胞的存在，因为微观毛细血管不容易与基质组织分离。因此，在分离过程的最早阶段达到尽可能高的纯度对于减少培养传代从而减少细胞老化至关重要。同样，最佳的隔离方案应提供尽可能高的纯HLMVECs产量。为了实现这些目标，基于先前描述的协议10，11，12，14?…
Discussion
血管内皮细胞在人体病理生理学中发挥的多种作用使这些细胞成为 体外 致病学和药理学研究不可或缺的工具。由于来自不同血管部位/器官的EC表现出特殊的特征和功能，因此来自感兴趣器官的健康和患病EC的可用性将是研究目的的理想选择。例如，HLMVEC对于肺部炎症的研究至关重要;因此，高产量、高纯度分离这些细胞的方法将有利于不同的研究领域。
分离HLMVEC的方法…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了意大利大学和研究部（前 60% 2021 年和 2022 年）对 R. P. 的资金支持，以及意大利囊性纤维化基金会 （FFC#23/2014） 和意大利卫生部 （L548/93） 对 M. R. 的赠款。
Materials
| 0.05% trypsin-EDTA 1X | GIBCO | 25300-054 | Used to detach cells from the culture plates |
| Anti CD31 Antibody, clone WM59 | Dako | M0823 | Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50 |
| Anti vWF Antibody | Thermo Fisher Scientific | MA5-14029 | Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100 |
| Autoclavable surgical scissors | Any | Used for chopping specimens | |
| Cell strainers 40 µm | Corning | 431750 | Used during the second filtration |
| Cell strainers 70 µm | Corning | 431751 | Using during the first filtration |
| Collagenase, Type 2 | Worthington | LS004177 | Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL |
| Conjugated anti CD31 Antibody | BD Biosciences | 555445 | Used for cell sorting (1:20 dilution) |
| Dulbecco′s Phosphate Buffered Saline (PBS) with CaCl2 and MgCl2 | Sigma-Aldrich | D8662 | Used for cell washing before medium change |
| Dulbecco′s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2 | Sigma-Aldrich | D8537 | Used for washing surgical specimens and cells before trypsinization |
| Endothelial Cell Growth Medium MV | PromoCell | C-22020 | HLMVEC growth medium |
| Fibronectin | Sigma-Aldrich | F0895 | Fibronectin from human plasma used for plate coating. Working concentration: 50 µg/mL |
| Gelatin from porcine skin, type A | Sigma-Aldrich | G2500 | Used for plate coating |
| Type A gelatin | Sigma-Aldrich | g-2500 | Gelatin from porcine skin used for plate coating. Working concentration: 1.5% |
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Cite This Article
Plebani, R., D’Alessandro, A., Lanuti, P., Simeone, P., Cinalli, M., Righi, I., Palleschi, A., Mucci, M., Marchisio, M., Cappabianca, F., Camera, M., Mucilli, F., Romano, M. Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples. J. Vis. Exp. (192), e64885, doi:10.3791/64885 (2023).