片上内皮炎症表型
Summary
应用光刻和蚀刻的PDMS制作的微流室探测与欧共体功能障碍和炎症相关的功能性结果。在有代表性的实验，能力差剪应力调节单核细胞粘附因子激活欧共体单层证明。
Abstract
动脉粥样硬化是potentiated高度的全身性炎症的状态，造成血管内皮功能障碍，有助于代谢异常。然而，早在血管内皮细胞的功能变化，意味着个人的风险程度是不直接评估临床治疗策略，以帮助指导。此外，调节局部血流动力学炎症导致动脉粥样硬化的非随机分布，但机制是难以界定， 在体内 。我们描述了实验室在一个芯片为基础的方法，以定量检测人血管内皮细胞（EC）和精确的流量条件下的单核细胞炎症活动的代谢扰动。软光刻技术的标准方法，用于1这些器件具有microfabricate 模仿血管微商会（VMMC）的 ，这必将直接培养欧共体膜。使用小体积，同时试剂的优势直接成像在接触到一个明确的剪切场欧共体膜炎症事件提供了一个平台。我们已成功地应用于这些设备，以探讨细胞因子，脂质3，4和RAGE诱导人类主动脉EC（HAEC）炎症。在这里，我们记录使用的检测单核细胞（THP-1）轧制和逮捕条件下差剪切特性及炎性细胞因子肿瘤坏死因子-α激活的HAEC单层VMMC。这类研究到atherosusceptibility提供的代谢危险因素在机械洞察力。
Protocol
1。细胞培养和基材的准备削减3英寸100×20 mm组织培养皿（BD猎鹰）使用车床的圆形基板。浸入70％乙醇消毒基板。 4毫升型胶原在室温下为1小时（100微克/毫升）在培养皿和大衣的地方，然后用4毫升1×PBS冲洗。 申请1毫升直接基板，暂停在6.5×10 5细胞/ ml和种子的人主动脉内皮细胞（HAEC，通过4-6）。放置在37℃，5％CO 2的孵化器，使细胞坚持以基材为1小时。 添加9…
Discussion
我们描述了微硅橡胶设备的使用片上，通过实时成像CAM的表达和单核细胞粘附内皮炎症型评估。我们的方法的一个主要优势在于能够量化细胞暴露炎症介质，如饮食中的脂肪和定义的水动力条件下的细胞因子，反映在动脉粥样硬化的血管剪应力的血管内皮功能障碍的相关成果。的VMMC方便使用少量的试剂或材料，可能是在有限的供应，并有增加的优点是价格低廉，可定制的，适合高吞吐量。
<p c…Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国立卫生研究院/ NHLBI赠款以R01 HL082689一西蒙斯科特和安东尼·G. Passerini。
Materials
| Item | Company | Catalogue Number |
| 100 x 20mm Petri Dishes | BD Falcon | 353003 |
| Ethanol 95% | EMD Chemicals | EX0290-1 |
| DPBS | Cellgro | 21-031-CV |
| Type I Rat Tail Derived Collagen | Gibco | A10483-01 |
| Human Aortic Endothelial Cells | Genlantis | PH30405A |
| Antibiotic-Antimycotic Solution | Invitrogen | 15240-062 |
| Endothelial BulletKit | Lonza | CC-4176 |
| Endothelial Basal Media-2 | Lonza | CC-3156 |
| 10 ml Syringes | BD Falcon | 309604 |
| Polyurethane tubing | Tygon | ABW0001 |
| Leibovitz-15 Media | Gibco | 11415-069 |
| Sylgard 184 Silicone Elastomer Base | Dow Corning | 184 |
| Sylgard 184 Silicone Elastomer Curing Agent | Dow Corning | 184 |
| SU8 Photoresist Master Wafer | UC Davis Pan Lab | N/A |
| Eclipse TE200 Inverted Microscope | Nikon | Eclipse TE200 |
| Syringe Pump | Harvard Apparatus | PHD2000 |
| 19 gauge hypodermic needle | Kendall | 8881 |
| THP-1 Monocytic Cell Line | ATCC | TIB-202 |
| HBSS (Hanks Buffered Saline Solution) with Ca2+/Mg2+ | Gibco | 14025-092 |
| Tumor Necrosis Factor Alpha (TNF-α) | R&D Systems | 210-TA-010 |
| Stromal Derived Factor – 1 (SDF-1) | R&D Systems | 350-NS-010 |
| RPMI 1640 | Cellgro | 10-040-CV |
| Human Serum Albumin (HSA) | ZLB Behring | NDC 0053-7680-32 |
Table 2. Specific reagents and equipment.
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Tags
On-Chip Endothelial Inflammatory PhenotypingAtherogenesisMetabolic AbnormalitiesSystemic InflammationEndothelial DysfunctionFunctional Changes In EndotheliumRisk AssessmentTherapeutic StrategyRegulation Of InflammationLocal HemodynamicsNon-random Spatial Distribution Of AtherosclerosisLab-on-a-chipQuantitatively Assay Metabolic PerturbationInflammatory Events In Endothelial CellsMonocytesFlow ConditionsSoft LithographyMicrofabricate Vascular Mimetic Microfluidic Chambers (VMMC)Cultured EC MonolayersImaging Inflammatory EventsShear FieldCytokine-induced InflammationLipid-induced InflammationRAGE-induced InflammationHuman Aortic EC (HAEC)Monocytic Cell Rolling And ArrestTNF-alpha
Citer Cet Article
DeVerse, J. S., Bailey, K. A., Foster, G. A., Mittal, V., Altman, S. M., Simon, S. I., Passerini, A. G. On-Chip Endothelial Inflammatory Phenotyping. J. Vis. Exp. (65), e4169, doi:10.3791/4169 (2012).