内皮化微流体研究在血液病的微血管相互作用
1Department of Pediatrics,Emory University School of Medicine, 2Wallace H. Coulter Department of Biomedical Engineering,Georgia Institute of Technology and Emory University, 3Aflac Cancer Center and Blood Disorders Service of Children’s Healthcare of Atlanta , 4Winship Cancer Institute of Emory University
Summary
方法文化在整个内部的微血管大小通道（<30微米）的微流体装置的三维表面的内皮细胞单层描述。这<em>在体外</em>微血管模型，使血细胞，内皮细胞，可溶性因子在血液系统疾病之间的生物物理相互作用的研究。
Abstract
在微细加工技术的进步使生产价格低廉，可重复的微流体系统进行生物和生化实验，在微，和nanoscales 1,2。此外，微流体也被专门用于定量分析血液和微血管的过程，因为他们有能力轻松地控制流体的动态环境和生物条件3-6。因此，研究人员最近利用微系统研究血细胞变形，血细胞聚集，微血管血流量，血细胞-内皮细胞相互作用6-13。然而，这些微系统并未包括培养的内皮细胞或较大比sizescale微血管的病理过程有关。与内皮细胞的微流体平台，准确地概括蜂窝，物理，和hemodyn微循环的的AMIC环境需要进一步的我们涉及微血管的血液系统疾病的生物物理基础病理生理学的理解。
在这里，我们报告的方法， 在体外微血管模型创建的“内皮化”，用一个简单的，单一的掩膜微细加工工艺标准的内皮细胞培养技术，研究发生在血液病的病理生物物理微血管相互作用结合。这种“微血管上的单芯片”的研究者提供了一个强大的检测，严格控制生物以及生物物理条件和操作使用标准的注射泵和明/荧光显微镜。如微循环血流动力学条件下，内皮细胞型，血细胞类型（S）和浓度（S），药物/抑菌浓度等参数都可以很容易地控制。正因为如此，我们的微提供方法定量调查疾病微血管流量由于改变细胞粘附，聚集和变形，无法与现有的检测能力受损的过程。
Protocol
1。内皮微器件的制备外面罩供应商提交的微流体装置的电脑辅助设计（CAD）绘图，创建一个光罩。使用面膜的钠钙玻璃上镀铬层组成。在这种情况下，微通道宽度为30微米。 15分钟的清洁与食人鱼裸硅晶片（10:1比例的硫酸和过氧化氢）和氢氟酸浸泡30秒。与去离子（DI）水冲洗约10秒。 使用旋转涂布机，旋转Microchem SU-8 2025到晶圆光阻到30微米的高度。对于SU-8 2025，3000转的转?…
Discussion
最适合我们的内皮化微装置系统结合， 在体内实验中使用时，其还原方法可能有助于阐明在人类和动物模型中观察到的血液进程的物理机制。此外，我们的系统也不是没有限制的。例如，我们的微通道截面是方形。虽然可以制作技术上圆形微10,11，我们选择使用更简单，更标准的制造过程，让其他研究人员，随时这个系统应用到自己的工作。此外，培养内皮细胞自然“轮出”有效?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢T.亨特，M.罗森布鲁斯，和他们的意见和有益的讨论林实验室。我们承认从G.微调，在佐治亚理工学院电子和纳米技术研究所的支持。由NIH资助的系列K08-HL093360 REAC奖，加州大学旧金山分校，美国国立卫生研究院奈米发展中心奖PN2EY018244，从亚特兰大儿童保健内皮细胞生物学中心的资金，为这项工作提供了资金支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| blunt point needle | OK International | 920050-TE | Precision TE needle 20 Gauge x 1/2″, pink |
| dextran | Sigma-Aldrich | 31392 | |
| Fibronectin | Sigma-Aldrich | F0895 | |
| Hole puncher (pin vise) | Technical Innovations | ||
| Human umbilical cord endothelial cells (HUVECs) | Lonza | CC-2519 | |
| Plasma cleaner | Plasma | PDC-326 | |
| Polydimethylsiloxane (PDMS) | Fisher Scientific | NC9285739 | Sylgard 184 Silicone Elastomer KIT |
| Sigmacote | Sigma-Aldrich | SL2 | |
| SU-8 2025 | Microchem | Y111069 | |
| SU-8 Developer | Microchem | Y020100 | |
| Syringe pump | Harvard Apparatus | 70-3008 | PHD-ULTRA |
| tubing(larger) | Cole-Parmer Instrument Company | 06418-02 | Tygonreg microbore tubing, 0.020″ ID x 0.060″ OD |
| tubing(smaller) | Cole-Parmer Instrument Company | 06417-11 | PTFE microbore tubing, 0.012″ ID x 0.030″ OD |
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Tags
MicrofluidicsEndothelialized MicrofluidicsMicrovascular InteractionsHematologic DiseasesMicrofabrication TechniquesBiological ExperimentsBiochemical ExperimentsMicro- And NanoscalesHematologic ProcessesBlood Cell DeformabilityBlood Cell AggregationMicrovascular Blood FlowBlood Cell-endothelial Cell InteractionsCultured Endothelial CellsMicrocirculationBiophysical Pathophysiology
Cite This Article
Myers, D. R., Sakurai, Y., Tran, R., Ahn, B., Hardy, E. T., Mannino, R., Kita, A., Tsai, M., Lam, W. A. Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases. J. Vis. Exp. (64), e3958, doi:10.3791/3958 (2012).