瓣膜内皮细胞的分离
Summary
我们提供了隔离和培养心脏瓣膜内皮细胞（VEC）的纯种群的方法。血管内皮细胞可以分离出了风口浪尖或单张两侧及紧随，底层的间质细胞（VIC）的隔离很简单。
Abstract
心脏瓣膜是全权负责维持心血管系统的单向血液流动。这些薄，纤维组织受到显着的机械应力，因为他们打开和关闭一个寿命超过数十亿倍。这些组织的令人难以置信的耐力是，由于居民瓣膜内皮细胞（VEC）和间质细胞（VIC）的不断响应当地的机械和生物信号的修复和改造。直到最近，我们开始认识到这些细胞的独特的行为，在体外实验中起到了关键作用。尤其是具有挑战性的是血管内皮细胞的分离和文化。必须特别小心使用的那一刻起，该组织是由主机，最后通过电镀删除。在这里，我们目前的直接隔离，边具体隔离，文化，和核查的VEC纯种群的协议。我们使用温柔棉签刮技术打跑只是表面细胞的酶消化。这些细胞，然后收集到管到一个沉淀离心。然后再悬浮颗粒和镀到培养瓶中，与Ⅰ型胶原基质预涂。血管内皮细胞的表型是由接触证实，抑制细菌的生长和PECAM1（CD31），如内皮细胞特异性标志物的表达，血管性血友病因子（vWF），α-平滑肌肌动蛋白（α- SMA）的表达阴性。血管内皮细胞的功能特点与乙酰化低密度脂蛋白水平高。与血管内皮细胞，血管内皮细胞有独特的能力，转化为间质细胞，它通常发生在胚胎阀形成 1 。在显着延长在体外培养后融合，这也可能发生，所以应达到或接近汇合通行。维也纳国际中心纯种群的VEC隔离后，然后可以很容易获得。
Protocol
1。制备在有盖的仪器高压灭菌托盘下列事项： 锯齿组织钳 – 对于处理单张组织组织剪（8厘米） – 修剪单张组织和尖棉签 – 从单张或风口浪尖上的内皮细胞层隔离设为无菌胶原酶溶液添加4.0克奶粉的DMEM 250毫升18MΩ的水。 添加碳酸氢钠1.11克。 新增（600 U / mL）的18万单位的胶原酶。 添加1％（3ML）青霉素/链霉素。 调整pH值?…
Discussion
技术隔离的困难和心脏瓣膜内皮细胞的培养纯人口已经受损的瓣膜生物学的理解。典型的隔离技术涉及的底层基础矩阵内皮胶粘剂债券2,3或化学分解酶消化。初步分离实验进行了定性评估，不同的解离剂和潜伏期。这些实验结果表明，EDTA（或胰蛋白酶- EDTA）的潜伏期长达60分钟撞出细胞不成功。然而，检索纯瓣膜内皮细胞（表1）的可用数量为5-10分钟collagense消化证明是有效的。其他酶解?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国家科学基金会职业奖，哈特韦尔基金会，和美国心脏协会（＃0830384N）的支持。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Dulbecco’s Modified Eagle Medium | Mediatech | 50-103-PB | ||
| Fetal Bovine Serum | Gibco | 26140 | ||
| Penicillin Streptomycin | Gibco | 15140-122 | ||
| 0.25% Trypsin-EDTA | Gibco | 25200 | ||
| Heparin Sodium Salt | Sigma-Aldrich | H4784-1G | ||
| Collagenase Type 2 | Worthington Biochemical | LS004176 | ||
| DPBS | Gibco | 21300-058 | ||
| Rat Tail Collagen | BD Biosciences | 354236 | ||
| Critical Swabs | VWR | 89031-270 | ||
| Sodium Bicarbonate | Sigma-Aldrich | 55761 | ||
| T25 Flasks | BD Biosciences | 353018 | ||
| T75 Flasks | BD Biosciences | 353136 | ||
| 24 Well Plate | Falcon | 353047 | ||
| 60×15 mm Dishes | VWR | 25384-092 | ||
| 60×15 Glass Dishes | VWR | 89000-310 | ||
| Paraffin Embedding Wax | Electron Microscopy Sciences | 19304-01 | ||
| Precision Glide Needles | BD Biosciences | 305165 | ||
| 500 mL Nalgene Filters | VWR | 73520-985 | ||
| 1L Nalgene Filters | VWR | 73520-986 | ||
| Tissue Forceps | Fine Science Tools | 11023-15 | ||
| FSC Tweezers #5 | Fine Science Tools | 11295-00 |
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Tags
IsolationValvular Endothelial CellsHeart ValvesUnidirectional Blood FlowCardiovascular SystemMechanical StressesValvular Endothelial CellsInterstitial CellsRepair And RemodelIn Vitro ExperimentationIsolation And CultureDirect IsolationSide Specific IsolationCultureVerification Of Pure PopulationsEnzymatic DigestionGentle Swab Scraping TechniqueSurface CellsCentrifuged Into A PelletResuspended And PlatedCollagen I MatrixVEC PhenotypeContact Inhibited GrowthEndothelial Specific Markers (PECAM1/CD31)Von Willebrand Factor (vWF)Alpha-smooth Muscle Actin (α-SMA)Functional Characteristics
Cite This Article
Gould, R. A., Butcher, J. T. Isolation of Valvular Endothelial Cells. J. Vis. Exp. (46), e2158, doi:10.3791/2158 (2010).