的系统分析<em>体外</em>细胞滚动使用多孔板微流控系统
1Division of Biomedical Engineering, Department of Medicine,Brigham and Women’s Hospital, 2Center for Regenerative Therapeutics,Brigham and Women’s Hospital, 3Harvard Medical School,Harvard University, 4Harvard Stem Cell Institute,Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering,Massachusetts Institute of Technology
Summary
本研究采用多孔板微流体系统，根据有关生理剪切流显著增加通量细胞滚动研究。定小区轧制的多步骤细胞归巢级联和细胞归巢的下列全身递送的细胞的患者的外源性群体的重要性的重要性，该系统提供了潜在的作为筛选平台，以提高基于细胞的疗法。
Abstract
细胞为基础的治疗的一个主要挑战是无法系统地针对大量活菌，高效率的兴趣组织下静脉或动脉内注射。因此，增加细胞归巢目前正在研究作为一项战略，以改善细胞疗法。细胞滚动对血管内皮是在细胞归巢的过程中的重要一步，可以使用平行板流室（PPFC） 在体外进行探测。然而，这是一个非常繁琐的，低通量的检测，控制不良的流动条件。相反，我们使用了一个多穴板的微流体系统，可以使蜂窝轧制性能研究在更高的吞吐量下精确控制的，生理学相关的剪切流动1,2。在本文中，我们表明HL-60（人早幼粒细胞白血病）上的P-和E-选择蛋白包被的表面，以及对细胞单层涂层表面细胞的轧制性能如何可以readilŸ检查。以更好地模拟炎性病症，所述微流体通道的表面上涂布的内皮细胞（EC），然后将其与肿瘤坏死因子-α（TNF-α）激活时，在动态条件下显著增加与HL-60细胞的相互作用。增强的吞吐量和集成的多参数分析的软件平台，允许的参数，如轧制速度和轧路径的快速分析，对评估体外细胞滚动属性重要的优点。使工程设计方法来影响细胞的滚动和归巢的快速，准确的分析，这个平台可以帮助推进外源性细胞为基础的治疗。
Introduction
一种在基于细胞的疗法的成功临床翻译的主要挑战是低效的交货或全身性输注的细胞靶向至期望的位点3,4。因此，人们不断寻求方法来改善细胞的归巢，并且特别是信元轧制，作为一个策略，以改善细胞疗法。细胞滚动血管在细胞归巢级联，经典的定义被招募到发病部位5白细胞的关键一步。这一步是由内皮细胞选择素之间的特异性相互作用制约， 即 P-和E-选择素（P-和E-SEL），和他们的白细胞5,6表面上柜台的配体。更好地了解和细胞归巢的提高效率，特别是轧制工序，是在寻求新的平台，以提高细胞为基础的治疗具有重要意义。迄今这已经通过使用平行平板流动腔（PPFCs），包括两个平板型实现ES与它们之间的垫圈，与位于上板的流入和流出端口，通过该细胞悬浮液是通过使用注射器泵7,8，9灌注。底板的表面可以涂覆有一个相关的细胞单层/基板和下剪切流灌注细胞和表面之间的相互作用，然后探讨7。然而，PPFC是低吞吐量，试剂消耗，以及相当繁琐的方法，用泡沫形成，泄漏，控制不佳的流量呈现主要缺点。
一种替代技术对传统PPFC是一个多穴板的微流体系统中，根据精确的，计算机控制的剪切流动，允许细胞分析的更高的吞吐量性能（比PPFCs高10倍），具有低试剂消耗1,10。细胞轧制实验内的微流体通道中进行的，它可以涂覆有细胞单层或工程衬底和成像USI吴显微镜，采用滚动属性，使用适当的软件易于分析。在本研究中，我们证明此多孔板的微流体系统的通过研究人早幼粒白血病在不同的表面（HL-60）细胞的滚动性能的能力。 HL-60轧像P-和E-SEL，以及对表达不同的轧制受体细胞单层，分析底物。此外，抗体（抗体）阻断被用于证明特定选择素介导的那些表面上的HL-60的滚动运动的直接参与。轧制实验，以增加吞吐量进行的，在稳定的剪切流动，以最少的试剂/细胞消耗，从而允许键滚动参数，如轧制速度，轧细胞的数目，并且滚道特性有效的分析。
Protocol
1。细胞培养人类早幼粒细胞白血病（HL-60）细胞培养HL-60细胞在75cm 2培养瓶中用15毫升的Iscove氏改良的Dulbecco氏培养基（IMDM），补充有20％（V / V）胎牛血清（FBS），1％（V / V）L-谷氨酰胺和1个％（体积/体积）青霉素 – 链霉素。 通过吸一半的细胞悬液体积和完整的IMDM培养基取代它改变媒体每3天。 为羧基二乙酸酯，琥珀酰亚胺酯（CFSE）染色，离心机的HL-60细胞悬…
Representative Results
HL-60细胞滚上的P-和E-选择素面，但不能在纤维连接蛋白 HL-60细胞被认为是金标准“辊”，因为它们表达多种归巢的配体，包括轧制配体的P-SEL糖蛋白配体-1（PSGL-1）和唾液酸-路易斯X（SLEX）的5,14（ 图1A ）。表面蛋白PSGL-1作为一个支架的四糖SLEX，介导特异性相互作用与P-和E-SEL，这是炎症5,6,15中上调的内皮。为了测试多穴板的微流体系统的功能，许多…
Discussion
其中外源性细胞为基础的治疗成功翻译的主要挑战是无法有效地提供细胞损伤和炎症，高效率的植入3的站点。细胞轧制代表在细胞归巢的过程中的一个关键步骤，促进血管壁上的细胞的减速，通过血管内皮细胞进入组织5最终导致其牢固粘附和轮回。轧制过程中对候选小区类型的更好的理解可能导致的技术的开发，提高细胞的归巢和朝着改善的基于细胞的疗法显著贡献。
<p class…Divulgaciones
The authors have nothing to disclose.
Acknowledgements
CHO-P细胞从芭芭拉FURIE博士（贝斯以色列女执事医疗中心，哈佛大学医学院）是一种礼物。到JMK这项工作是由一个Movember的前列腺癌基金会挑战奖，以JMK也支持这部分工作为卫生补助HL095722研究所
Materials
| Cells | |||
| Human Lung Microvascular Endothelial Cells | Lonza | CC-2527 | |
| P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P) | Kind gift by Dr. Barbara Furie11,12 | ||
| HL-60 Cells | ATCC | CCL-240 | |
| [header] | |||
| Cell Culture Reagents | |||
| Endothelial Basal Medium | Lonza | CC-3156 | |
| EBM-2 Media | Lonza | CC-3156 | |
| Endothelial Basal Medium Supplements | Lonza | CC-4147 | |
| EGM-2 MV SingleQuots | Lonza | CC-4147 | |
| IMDM – Iscove's Modified Dulbecco's Medium 1x | Gibco | 12440 | |
| F-12 (1x) Nutrient Mixture (Ham) | Gibco | 11765-054 | |
| Penicillin Streptomycin (P/S) | Gibco | 15140 | |
| L-Glutamine (L/G) 200 mM | Gibco | 25030 | |
| Fetal Bovine Serum (FBS) | Atlanta Biologicals | Sa550 | |
| Petri Dishes | BD Falcon | BD-353003 | |
| 100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene | |||
| Centrifuge Tubes (15 ml polypropylene conical tubes) | MedSupply Partners | TC1500 | |
| T75 Flasks | BD Falcon | 353136 | |
| Gelatin Solution (2%) | Sigma | G1393 | |
| dPBS (without calcium chloride and magnesium chloride) | Sigma | D8537 | |
| Trypsin-EDTA Solution (10x) | Sigma | T4174 | |
| [header] | |||
| Antibodies | |||
| Anti-hE-Selectin/CD62E | R&D Systems | BBA21 | |
| FITC Conjugated Mouse IgG1 | R&D Systems | BBA21 | |
| Anti-hP-Selectin | R&D Systems | BBA34 | |
| FITC Conjugated Mouse IgG1 | R&D Systems | BBA34 | |
| FITC Mouse IgG1 κ Isotype Control | BD Bioscience | 555748 | |
| Anti-SLeX /CD15s Ab, Clone: 5F18 | Santa Cruz | SC70545 | |
| FITC Conjugated | Santa Cruz | SC70545 | |
| Normal Mouse IgM-FITC Isotype Control | Santa Cruz | SC2859 | |
| PE Mouse Anti-Human CD162, Clone: KPL-1 | BD Pharmingen | 556055 | |
| PE Mouse IgG1 k Isotype Control | BD Pharmingen | 550617 | |
| Anti-P-Selectin Ab (AK4) | Santa Cruz | SC19996 | |
| Anti-E-Selectin Ab, Clone P2H3 | Millipore | MAB2150 | |
| Mouse IgG1 Isotype Control | Santa Cruz | SC3877 | |
| [header] | |||
| Other Reagents | |||
| Recombinant Human TNF-alpha | PeproTech | 300-01A | |
| Cell Trace CFSE Cell Proliferation Kit – For Flow Cytometry | Invitrogen | C34554 | |
| Human P-selectin-FC recombinant protein | R&D Systems | 137-PS-050 | |
| Human E-selectin-FC recombinant protein | R&D Systems | 724-ES-100 | |
| Fibronectin Human, Plasma | Invitrogen | 33016-015 | |
| [header] | |||
| Equipment | |||
| Bioflux 1000 | Fluxion Biosciences | Bioflux Montage was the software used to run the experiments and analyze the data | |
| BioFlux 48-well plates | Fluxion Biosciences | ||
| BD Accuri C6 Flow Cytometer | BD Bioscience | CFlow Plus was the software used to run the experiments and analyze the data | |
| Nikon Eclipse Ti-S | Nikon | ||
| CoolSnap HQ2 CCD camera | Photometrics |
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Citar este artículo
Levy, O., Anandakumaran, P., Ngai, J., Karnik, R., Karp, J. M. Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System. J. Vis. Exp. (80), e50866, doi:10.3791/50866 (2013).