在小型组织培养体系培养老鼠心脏瓣膜
Summary
Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.
Abstract
Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure.
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive.
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.
Introduction
心脏瓣膜疾病是发病率和死亡率在西方世界的一个主要原因;其患病率随着年龄的增加，它会影响人口75年的10％以上和更老1。心脏的全身性部分的阀门，主动脉和二尖瓣，大多是受影响的。心脏瓣膜病的特征是所述阀的高度组织的结构，这导致的机械性能2变更的损失。结构完整性，因此对阀的功能是至关重要的。
所述瓣膜的瓣叶被包括瓣膜间质细胞（VIC），瓣膜内皮细胞（VEC），和细胞外基质，这是高度组织在一个分层图案3,4的。所述的VIC负责对ECM合成，降解和组织。因素从血液，内皮细胞发出或居住在编排上其功能的VIC的ECM行为。此外，机械力造成层流或振荡剪切应力，影响的VIC 5的行为压缩或拉伸应力在心动周期中作用于单张。
为了理解如何在阀的结构被调整，首先必须理解的VIC到心脏周期期间所经历的刺激的多样化如何作出反应。 在体外研究已经非常翔实有关瓣膜细胞的特征和能力。这些细胞在体外的反应，但是，可能不总是精确地模仿体内响应6;例如，VIC的对刺激的响应是依赖于内皮的存在和ECM 组合物5。此外，瓣膜细胞对刺激的响应取决于其特定的位置，在小叶7。除了生化刺激，瓣膜细胞的行为是通过作用Ø机械力确定n个阀 8。阀的各区域进行其自己的特定的一组血液动力学应力。尽管目前的体外模型表明，机械力的作用是瓣膜结构 5的重要决定因素，相关的机制仍不清楚。虽然体内模型提供了深入了解潜在的心脏瓣膜发展9,10的分子机制，见解成人瓣膜生物学仍然是难以捉摸的。
因此，开发了离体的流动的模型，其中该心脏瓣膜可以在其在心脏中自然位置的时间11长时间进行培养。这具有的优点是，阀保持在其天然构型和的VIC经历相同的环境作为体内，使得的VIC响应的刺激尽可能自然。此外，阀门在其自然位置在心脏的文化有利于使各瓣膜区域相关的血液动力学应力。在此体外模型，也就是说，微型组织培养系统（MTCS），所述阀可进行不同的生物化学和血液动力学刺激允许其在心脏瓣膜重塑作用的调查。
Protocol
Representative Results
Discussion
在培养该心脏小鼠阀关键步骤包括使来自鼠标切除心脏的和结扎在灌注腔尽可能短，以确保在垂直于阀的可行性的针和结扎之间的时间，以确保流动的正确方向。此外，检查在灌注腔结扎后的流量不中确保适当的插入和针结扎。关键是要保持无菌培养和防止气泡在油管，这有可能被困在心脏阻碍的流动。
用于心脏的灌注介质的总体积是45毫升（注意，即通过心脏灌注介质是不…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.
Materials
| Dulbecco’s Modified Eagle Medium | life technolgies | 10569-010 | |
| Fetal Bovine Serum | life technolgies | 26140 | |
| Insulin-Transferrin-Selenium | life technolgies | 41400-045 | |
| Antibiotic-Antimycotic | life technolgies | 15240-06 | |
| Silk 7-0 | Ethicon | 768G | |
| 100 mm culture dish | Greiner bio-one | 664160 | |
| 50 ml tube | Greiner bio-one | 227261 | |
| 5 ml syringe | BD | 309649 | |
| 21 G needle | BD | 304432 | |
| Heparin | LEO | 012866-08 | |
| Forceps | Fine Science Tools | 11295-00 | |
| Micro Scissors, Economy, Vannas-type | Tedpella | 1346 | |
| Silicon tubing | Thermo Scientific | 8060-0020 | I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm |
| Silicon tubing for pump | Masterflex | 96400-13 | I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm |
| Biocompatible glue (Histoacryl) | B. Braun | 1050071 | |
| precision vaporizer | Dräger | Vapor 200 | |
| peristaltic roller pump | Masterflex | 7521-35 | |
| Easy-load pump head | Masterflex | 7518-00 | |
| Flow chamber | see Lieber et al., 2010 | ||
| Bubble trap | see Lieber et al., 2010 |
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