Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels
Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels
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DOI：
10.1007/978-1-4939-3628-1_4
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发表时间：
2016-01-01
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影响因子：
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通讯作者：
Hewett, Peter W.
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作者：
Hewett, Peter W.
不同血管床的内皮细胞表现出高度的表型异质性。内皮细胞（EC）可以通过机械去除或胶原酶消化很容易地从大血管中收获。特别是，人类脐静脉由于其广泛的可用性而被使用，而对其衍生的内皮细胞的研究无疑大大提高了我们对血管生物学的认识。然而，身体的大部分内皮细胞（约95%）形成了微血管，正是这些细胞提供了血液和组织之间的界面，在新血管的形成中起着关键作用。这导致了从不同组织中分离微血管内皮细胞（MEC）的技术的建立，以提供更多与血管生成和内皮细胞功能生理相关的体外模型。在本章中，描述了使用涂有抗pecam -1 （CD31）抗体（PECA-beads）的超顺磁珠（Dynabeads）培养来自人脂肪组织的mec，以及用于表征它们的标准方法。脂肪组织是MECs的理想来源，因为它主要由脂肪细胞组成，具有非常丰富的微血管，易于分解。此外，它可以在整形手术过程中大量获得。乳房缩小成形术或腹部成形术获得的脂肪首先剥离结缔组织，切碎，并进行II型胶原酶消化。通过离心除去脂肪细胞，得到微血管丰富的颗粒，用胰蛋白酶/EDTA溶液进一步分解。过滤去除结缔组织碎片后，将颗粒与peca -珠和微血管碎片/ECs孵育，并用磁铁洗涤和收获。此外，本文还描述了将这一基本技术应用于人肺和胃mec的分离，以及制备大血管内皮细胞的常用方法。
The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells.