Umbilical cords were collected with the assistance of the Birmingham Women's Health Care NHS Trust. HMM was supported by an Arthritis Research UK Career Development Fellowship (19899) and Systems Science for Health, University of Birmingham (5212).

<ol>
	<li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+on+endothelium+for+lymphocyte+recirculation+and+leukocyte+emigration.">Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.</a> Ann. Rev. Physiol. 57, 827-872 (1995).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nature reviews. Immunology. 7 (9), 678-689 (2007).</li><li>McGettrick, H. M., Butler, L. M., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stroma+as+a+regulator+of+leukocyte+recruitment+in+inflammation.">Tissue stroma as a regulator of leukocyte recruitment in inflammation.</a> Journal of leukocyte biology. 91 (3), 385-400 (2012).</li><li>Serhan, C. N., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+the+beginning+programs+the+end.">Resolution of inflammation: the beginning programs the end.</a> Nature immunology. 6 (12), 1191-1197 (2005).</li><li>Schmidt, S., Moser, M., Sperandio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+leukocyte+recruitment+and+its+deficiencies.">The molecular basis of leukocyte recruitment and its deficiencies.</a> Molecular immunology. 55, 49-58 (2013).</li><li>Luu, N. T., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Ability+of+Exogenous+Chemotactic+Agents+to+Disrupt+Transendothelial+Migration+of+Flowing+Neutrophils.">Differential Ability of Exogenous Chemotactic Agents to Disrupt Transendothelial Migration of Flowing Neutrophils.</a> The Journal of Immunology. 164 (11), 5961-5969 (2000).</li><li>Smith, C. W., Rothlein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+an+endothelial+determinant+for+CD+18-dependent+human+neutrophil+adherence+and+transendothelial+migration.">Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration.</a> The Journal of clinical investigation. 82 (5), 1745-1756 (1988).</li><li>Luscinskas, F. W., Brock, A. F., Arnaout, M. A., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-leukocyte+adhesion+molecule-1-dependent+and+leukocyte+(CD11/CD18)-dependent+mechanisms+contribute+to+polymorphonuclear+leukocyte+adhesion+to+cytokine-activated+human+vascular+endothelium.">Endothelial-leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium.</a> J. Immunol. 142 (7), (1989).</li><li>Bahra, P., Rainger, G. E., Wautier, J. L., Nguyet-Thin, L., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Each+step+during+transendothelial+migration+of+flowing+neutrophils+is+regulated+by+the+stimulatory+concentration+of+tumour+necrosis+factor-alpha.">Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha.</a> Cell adhesion and communication. 6 (6), 491-501 (1998).</li><li>Piali, L., Weber, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+receptor+CXCR3+mediates+rapid+and+shear-resistant+adhesion-induction+of+effector+T+lymphocytes+by+the+chemokines+IP10+and+Mig.">The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig.</a> European journal of immunology. 28 (3), 961-972 (1998).</li><li>McGettrick, H. M., Smith, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+from+different+sites+may+promote+or+inhibit+recruitment+of+flowing+lymphocytes+by+endothelial+cells.">Fibroblasts from different sites may promote or inhibit recruitment of flowing lymphocytes by endothelial cells.</a> European journal of immunology. 39 (1), 113-125 (2009).</li><li>McGettrick, H. M., Hunter, K., Moss, P. a., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+kinetics+of+migrating+T+cells+suggest+active+retention+by+endothelial+cells+with+continual+bidirectional+migration.">Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration.</a> Journal of leukocyte biology. 85 (1), 98-107 (2009).</li><li>McGettrick, H. M., Buckley, C. D., Filer, A., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+differentially+regulate+neutrophil+and+lymphocyte+recruitment+through+the+endothelium.">Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium.</a> Immunology. 131 (3), 357-370 (2010).</li><li>Tull, S. P., Yates, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omega-3+Fatty+acids+and+inflammation:+novel+interactions+reveal+a+new+step+in+neutrophil+recruitment.">Omega-3 Fatty acids and inflammation: novel interactions reveal a new step in neutrophil recruitment.</a> PLoS biology. 7 (8), e1000177 (2009).</li><li>Ahmed, S. R., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+D2+regulates+CD4++memory+T+cell+trafficking+across+blood+vascular+endothelium+and+primes+these+cells+for+clearance+across+lymphatic+endothelium.">Prostaglandin D2 regulates CD4+ memory T cell trafficking across blood vascular endothelium and primes these cells for clearance across lymphatic endothelium.</a> Journal of immunology. 187 (3), 1432-1439 (2011).</li><li>Bradfield, P. F., Amft, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+fibroblast-like+synoviocytes+overexpress+the+chemokine+stromal+cell-derived+factor+1+(CXCL12),+which+supports+distinct+patterns+and+rates+of+CD4++and+CD8++T+cell+migration+within+synovial+tissue.">Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue.</a> Arthritis and rheumatism. 48 (9), 2472-2482 (2003).</li><li>Filer, A., Parsonage, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+survival+of+leukocyte+subsets+mediated+by+synovial,+bone+marrow,+and+skin+fibroblasts:+site-specific+versus+activation-dependent+survival+of+T+cells+and+neutrophils.">Differential survival of leukocyte subsets mediated by synovial, bone marrow, and skin fibroblasts: site-specific versus activation-dependent survival of T cells and neutrophils.</a> Arthritis and rheumatism. 54 (7), 2096-2108 (2006).</li><li>Lally, F., Smith, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mechanism+of+neutrophil+recruitment+in+a+coculture+model+of+the+rheumatoid+synovium.">A novel mechanism of neutrophil recruitment in a coculture model of the rheumatoid synovium.</a> Arthritis and rheumatism. 52 (11), 3460-3490 (2005).</li><li>Chakravorty, S. J., McGettrick, H. M., Butler, L. M., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro.+model+for+analysing+neutrophil+migration+into+and+away+from+the+sub-endothelial+space:+Roles+of+flow+and+CD31.">An in vitro. model for analysing neutrophil migration into and away from the sub-endothelial space: Roles of flow and CD31.</a> Biorheology. 43 (1), 71-82 (2006).</li><li>Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Pathology+of+Atherosclerosis+Smooth+Muscle+Cells+Prime+Cocultured+Endothelial+Cells+for+Enhanced+Leukocyte+Adhesion.">Cellular Pathology of Atherosclerosis Smooth Muscle Cells Prime Cocultured Endothelial Cells for Enhanced Leukocyte Adhesion.</a> Circulation Research. 88 (6), 615-622 (2001).</li><li>Kuravi, S. J., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podocytes+regulate+neutrophil+recruitment+by+glomerular+endothelial+cells+via+IL-6-mediated+crosstalk.">Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk.</a> Journal of immunology. 193 (1), 234-243 (2004).</li><li>Parsonage, G., Filer, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stromal+address+code+defined+by+fibroblasts.">A stromal address code defined by fibroblasts.</a> Trends in immunology. 26 (3), 150-156 (2005).</li><li>Luu, N. T., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+mesenchymal+stem+cells+and+endothelial+cells+leads+to+downregulation+of+cytokine-induced+leukocyte+recruitment.">Crosstalk between mesenchymal stem cells and endothelial cells leads to downregulation of cytokine-induced leukocyte recruitment.</a> Stem cells. 31 (12), 2690-2702 (2013).</li><li>Bevilacqua, M. P., Nelson, R. M., Mannori, G., Cecconi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-leukocyte+adhesion+molecules+in+human+disease.">Endothelial-leukocyte adhesion molecules in human disease.</a> Annual review of medicine. 45, 361-378 (1994).</li><li>Stanness, K. A., Beatty, P. G., Ochs, H. D., Harlan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endothelial+cell+surface+factor(s)+induced+in+vitro.">An endothelial cell surface factor(s) induced in vitro.</a> 136 (12), 4548-4553 (1986).</li><li>Burton, V. J., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+of+migrating+leukocytes+by+the+basement+membrane+deposited+by+endothelial+cells+in+long-term+culture.">Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture.</a> Experimental cell research. 317 (3), 276-292 (2011).</li><li>Luu, N. T., Rainger, G. E., Buckley, C. D., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD31+Regulates+Direction+and+Rate+of+Neutrophil+Migration+over+and+under+Endothelial+Cells.">CD31 Regulates Direction and Rate of Neutrophil Migration over and under Endothelial Cells.</a> Journal of Vascular Research. 40 (5), 467-479 (2003).</li><li>McGettrick, H. M., Buckley, C. D., Ed Rainger, G., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stromal+cells+on+lymphocyte+adhesion+and+migration+on+endothelial+cells.">Influence of stromal cells on lymphocyte adhesion and migration on endothelial cells.</a> Methods in molecular biology. 616, 49-68 (2010).</li><li>Butler, L. M., McGettrick, H. M., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+and+dynamic+assays+of+cell+adhesion+relevant+to+the+vasculature.">Static and dynamic assays of cell adhesion relevant to the vasculature.</a> Methods in molecular biology. 467, 211-228 (2009).</li><li>Jeffery, H. C., Buckley, C. D., Moss, P., Rainger, G. E., Nash, G. B., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+effects+of+stromal+cells+on+the+migration+of+lymphocytes+into+and+through+inflamed+tissue+using+3-D+culture+models.">Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models.</a> Journal of immunological methods. 400-401, 45-57 (2013).</li></ol>

The authors declare that they have no conflicts of interest.

在这里，我们介绍两个体外 "血管"的模型来研究由内皮细胞发炎循环中性粒细胞的招募。这些模型的一个主要优点是分析中的白细胞粘附级联的每个步骤，以便，如将发生在体内的能力。我们先前观察到的剂量依赖性增加中性粒细胞粘附和轮回通过TNFα刺激的EC 9,29。我们还描述了这两种模式如何适应学习基质细胞对白细胞招聘的影响。这里，MSC共培养用乳油在Ibidi的MicroSlide或多孔过滤器的相对侧。这使得这两种类型的细胞，以相互通信，从而改变彼此的表型和响应。我们在这里显示，在以往的研究23，即MSC的存在抑制中性粒细胞粘附到TNFα刺激的EC。这表明，基质细胞修改EC响应细胞因子，并随后改变循环中的白细胞招聘。 除了上面描述的模式，Cellix生物芯片，Bioflux板和Glyotech平行平板流动腔室可商，对于培养的内皮和观察招募提供一种表面可用流路系统。在所有系统中，特定的参数，应考虑而建立内皮培养物并进行基于流的密合性试验中，其中一些在下面突出并在以往的报告30,31。任何抗炎剂，如氢化可的松，可能影响细胞因子应答应当由用于培养和测定18,29的持续时间的介质被省略。当使用Ibidi保证的MicroSlide有欧盟的培养过程中无气泡存在于流道，因为这些会破坏EC单层。内皮单层的完整性应该确认之前cytokiNE-治疗，嗜中性粒细胞将结合在了BSA已涂的MicroSlide /过滤器单层的空白。此外，还必须确保EC在整个细胞因子刺激维持在37℃，因为TNFα是温度敏感的，只有在37℃下的最大效果。对于流量检测本身，选择合适的洗涤液中，我们通常使用PBSA短期分析（少于30分钟）和M199培养基中添加BSA（0.15％），更长的分析（1 - 48小时）30,31。最后确保有在测定期间没有气泡存在于所述流动通道，因为这会破坏流率，损害欧共体单层和激活嗜中性粒细胞粘附。 一此处所述的多细胞的体外模型的主要优点是它们的复制EC和基质细胞之间的体内相互作用的能力。它是难以分离的体内特异性基质成分的作用并修改它们以可控的方式。在我们的模型中，基质细胞可以被操纵以阐明它们与EC如何进行通信，并影响炎症过程的健康和疾病。例如，使用siRNA的技术，我们以前曾表明，生产的IL-6由MSC在共培养物所需的其免疫抑制作用23。每个模型可以用于解决特定问题，即，在白细胞募集组织驻留基质细胞（基于过滤器的模型）或治疗性施用基质细胞（Ibidi microslides和其它市售的系统）的影响。在这两种情况下，我们滴定不同的基质细胞类型，以确保其可行性，并建立基质细胞的一个合适的比例，EC评估招聘15,25的影响。同样培养基必须与并入模型中的每个细胞类型相容。在我们手中共培养物中的基质细胞米典型地edium 11,18,23,31。 使用基于过滤器的模型，我们以前曾表明，各种基质细胞调制EC中以支持粘附和白细胞的迁移以组织特异性方式的能力，并且这些效果变得改变在慢性病3。这导致了基质细胞建立的组织特异性"地址代码"，它积极调节白细胞的募集至发炎的组织24的概念。这些模型是专门设计来检查招募的初始阶段的很详细，但无法研究内皮下空间内的后续的迁移（ 即，远离内皮进入组织）。多细胞，多层次的三维结构，如静态胶原凝胶实验12,32会更适合学习后者的招聘阶段。 我们基于流的黏附模式具有很强的通用性。我们有DEScribed其在中性粒细胞募集的上下文中的使用，但其它的白细胞亚群可以以类似的方式进行研究。我们还使用了Ibidi系统的MicroSlide调查循环MSC通过EC 23的招聘，以及是否发生过同样的附着力级联报道了白细胞。同样地，这些模型可用于研究招募和转移性的肿瘤细胞系的结合，以及它们对内皮反应和白细胞募集后续影响。可替代地，基于过滤器的模型可以适合于从健康组织13,21和疾病11,18,20的部位结合的不同类型的基质细胞（ 例如，成纤维细胞，足细胞，平滑肌细胞）的。这将使得能够组织特异性作用于对EC和/或白细胞的水平调节途径的研究。在所有情况下，在一个范围内的疾病状况正常调节过程的破坏可以被检查，以确定柯ÿ调节介质（如IL-6和TGF-β）和潜在的新的治疗靶点。在慢性炎症的情况下，这些药物可以用来关闭招聘过程，而在癌症生物学人能想象他们的使用，以打开招聘靶向肿瘤。

炎症是对微生物感染或组织损伤的保护性反应，需要白细胞条目的紧调节进入和退出从发炎组织，以允许第1,2。内皮细胞（EC），该行的血管，循环白细胞和组织驻留基质细胞之间的串扰是用于协调该过程3是必不可少的。然而，白细胞不受控制招聘和他们的无效间隙支撑的慢性炎症性疾病4的开发。我们目前在健康和疾病白细胞募集的理解是不完整的，还需要更可靠的模型来分析该过程。 通过血管EC的毛细血管后静脉支持从血液内白血球的招聘机制已经很好地描述1,2,5。循环白细胞是通过专门的受体（ 例如，VCAM-1，E-选择素，P-选择素）捕获该正在上调对发炎内皮。这些短暂的相互作用使白细胞与表面结合趋化因子和脂质衍生的介质（或内皮细胞间质或起源）的激活白细胞6 11表达整合互动。这反过来又稳定穿过内皮和进入组织12- 15粘附和驱动器迁移。在组织，招募白细胞受到影响他们的运动，功能和存活16,17基质衍生剂。越来越多的证据有力地表明，信号在招聘过程中条件的白细胞在接下来的每个阶段获得。然而，我们的白细胞招聘的认识仍然不完整，很少有人知道组织内的组件整形白细胞的运动。 在伯明翰，我们已经开发出多种体外 "血管"的车型，从研究白细胞招聘流9,18,19。我们现在明白了，白细胞募集血管EC充当即时监管响应变化的局部微环境。具体地，组织驻留基质细胞可以通过与邻近血管EC交谈来影响在招募3的作用积极调节炎症反应，这部分。我们以前曾表明，各种基质细胞调制EC中以支持粘附和白细胞的迁移以组织特异性方式的能力，而且，这些效果变得改变在慢性疾病13,16,20,21。因此，基质细胞建立组织&#39;地址代码&#39;定义每个炎性应答22的上下文中。最近，我们已经表明，骨髓来源的MSC（BMMSC）有力地下调欧盟的细胞因子的反应，导致减少在这两个中性粒细胞的招募和淋巴细胞23。 该机制GOVerning招聘阐明在体外已大量使用纳入一个单一的细胞类型（ 例如，EC）或蛋白质隔离检测。然而，这些研究并没有考虑到局部组织环境的影响（ 即，基质细胞的存在下）对招募白细胞及其随后迁移进入组织。在这里，我们描述了两个基于流的方法，其中的基质细胞，特异性间质干细胞（MSC），是共培养EC 23。这种模型允许我们检查基质细胞对内皮反应的影响，特别是它们从流支持白细胞募集的能力。

<table><tbody><tr><td>Collagenase Type Ia</td><td>Sigma</td><td>C2674</td><td>Dilute in 10 ml PBS to get a final concentration of 10 mg/ml. Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma</td><td>D8662</td><td>With calcium and magnesium chloride. Keep sterile and store at RT.</td></tr><tr><td>1X Medium M199</td><td>Gibco</td><td>31150-022</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Gentamicin sulphate</td><td>Sigma</td><td>G1397</td><td>Store at 4 &amp;deg;C. Add to M199 500 ml bottle.</td></tr><tr><td>Human epidermal growth factor</td><td>Sigma</td><td>E9644</td><td>Store at -20 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Sigma</td><td>F9665</td><td>FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56 &amp;deg;C. Store in 10 ml aliquots at -20 &amp;deg;C.</td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290-026</td><td>Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0135</td><td>Stock is in ethanol. Store at -20 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>Collagenase Type II</td><td>Sigma</td><td>C6885</td><td>Dilute stock in PBS to a final concentration of 100 mg/ml. Store at -20 &amp;deg;C in 100 &amp;micro;l aliquots.</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3631</td><td>Dilute stock in PBS to a final concentration of 20,000 U/ml. Store at -20 &amp;deg;C in 100 &amp;micro;l aliquots.</td></tr><tr><td>100 &amp;micro;m cell strainer for 50 ml centifuge tube</td><td>Scientific Lab Supplies (SLS)</td><td>352360</td><td>Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.</td></tr><tr><td>DMEM low glucose</td><td>Biosera</td><td>LM-D1102/500</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Penicillin/Streptomycin mix</td><td>Sigma</td><td>P4333</td><td>Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>25 cc tissue culture flask</td><td>SLS</td><td>353109</td><td></td></tr><tr><td>75 cc tissue culture flask</td><td>SLS</td><td>353136</td><td></td></tr><tr><td>Bone marrow mesenchymal stem cells vial</td><td>Lonza</td><td>PT-2501</td><td>Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.</td></tr><tr><td>Mesenchymal stem cell growth medium (MSCGM)</td><td>Lonza</td><td>PT-3001</td><td>Warm in 37 &amp;deg;C water bath before use. For Cell Tracker Green staining use medium without FCS.</td></tr><tr><td>EDTA (0.02%) solution</td><td>Sigma</td><td>E8008</td><td>Store at 4 &amp;deg;C. Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Trypsin solution</td><td>Sigma</td><td>T4424</td><td>Store at -20 &amp;deg;C in 2 ml aliquots. Thaw at RT and use immediately.</td></tr><tr><td>Cryovials</td><td>Greiner bio-one</td><td>2019-02</td><td>Keep on ice before adding before adding cell suspension.</td></tr><tr><td>Mr. Frosty Freezing Container</td><td>Nalgene</td><td>5100-0001</td><td>Store at RT. When adding cryovials with cells store at -80 &amp;deg;C for 24 hr before transfering cells to liquid nitrogen.</td></tr><tr><td>Ibidi u-Slide VI (0.4), T/C treated, sterile</td><td>Ibidi</td><td>IB-80606</td><td>Alternative models include glass capillaries, Cellix Biochips (www.cellixltd.com), BioFlux Plates (www.fluxionbio.com/bioflux/) and GlycoTech parallel plate flow chambers (http://www.glycotech.com/apparatus/parallel.html).</td></tr><tr><td>Cell tracker green dye</td><td>Life technologies</td><td>C2925</td><td>Store in 5 &amp;micro;l aliquots at -20 &amp;deg;C. Dilute in 5 ml prewarmed (at 37 &amp;deg;C) MSCGM.</td></tr><tr><td>Cell counting chambers</td><td>Nexcelom</td><td>SD-100</td><td>Alternatively a haemocytometer can be used.</td></tr><tr><td>Cellometer auto T4 cell counter</td><td>Nexcelom</td><td>Auto T4-203-0238</td><td></td></tr><tr><td>Tumor necrosis factor &amp;alpha; (TNF&amp;alpha;)</td><td>R&amp;amp;D Systems</td><td>210-TA-100</td><td>Dilute stock in PBS to a final concentration of 100,000 U/ml. Store at -80 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>6-well, 0.4 &amp;micro;m PET Transwell filters</td><td>SLS</td><td>353090</td><td></td></tr><tr><td>K2-EDTA in 10ml tubes</td><td>Sarstedt</td><td></td><td>Store at RT.</td></tr><tr><td>Histopaque 1119</td><td>Sigma</td><td>11191</td><td>Store at 4 &amp;deg;C. Warm to RT before use.</td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>10771</td><td>Store at 4 &amp;deg;C. Warm to RT before use.</td></tr><tr><td>10 ml round bottomed tube</td><td>Appleton Woods</td><td>SC211 142 AS</td><td></td></tr><tr><td>7.5% BSA Fraction V solution</td><td>Life technologies</td><td>15260-037</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>20 ml Plastipak syringes</td><td>BD falcon</td><td>300613</td><td></td></tr><tr><td>5 ml Plastipak syringes</td><td>BD falcon</td><td>302187</td><td></td></tr><tr><td>2 ml Plastipak syringes</td><td>BD falcon</td><td>300185</td><td></td></tr><tr><td>3M hypo-allergenic surgical tape 9 m x 2.5 cm</td><td>Micropore</td><td>1530-1</td><td>Use to secure the syringe tap onto the wall of the perspex chamber.</td></tr><tr><td>Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3 mm (thin tubing)</td><td>Fisher Scientific</td><td>FB68854</td><td>Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.</td></tr><tr><td>Silicon rubber tubing ID/OD of 2/4 mm (thick tubing)</td><td>Fisher Scientific</td><td>FB68855</td><td></td></tr><tr><td>Portex Blue Line Manometer tubing</td><td>Smiths</td><td>200/495/200</td><td>Tubing leading to the syringe pump.</td></tr><tr><td>3-way stopcock</td><td>BOC Ohmeda AB</td><td></td><td></td></tr><tr><td>Glass 50 ml syringe for pump</td><td>Popper Micromate</td><td>550962</td><td>Must be primed prior to use by removing any air bubbles.</td></tr><tr><td>Glass coverslip</td><td>Raymond A Lamb</td><td></td><td>26 x 76 mm coverslips made to order. Lot number 2440980.</td></tr><tr><td>Parafilm gasket</td><td>American National Can Company</td><td></td><td>Cut a 26 x 76 mm piece of parafilm using an aluminium template and cut a 20 x 4 mm slot into it using a scalpel 10a. Gasket thickness is approximately 133 &amp;micro;m.</td></tr><tr><td>Two perspex parallel plates</td><td>Wolfson Applied Technology Laboratory</td><td></td><td>Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.</td></tr><tr><td>Electronic 3-way microvalve with min. dead space</td><td>Lee Products Ltd.</td><td>LFYA1226032H</td><td>Electronically connected to a 12 volt DC power supply.</td></tr><tr><td>Syringe pump for infusion/withdrawal (PHD2000)</td><td>Harvard Apparatus</td><td>70-2001</td><td>Set the diameter to 29 mm and refill (flow) rate.</td></tr><tr><td>L-shaped connector</td><td>Labhut</td><td>LE876</td><td>To attach to the inlet and outlet ports onto the Ibidi microslide channel.</td></tr><tr><td>Video camera</td><td>Qimaging</td><td>01-QIC-F-M-12-C</td><td>Connected to a computer which enables digitall videos to be recorded.</td></tr><tr><td>Image-Pro Plus 7.0</td><td>Media Cybernetics</td><td>41N70000-61592</td><td>For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.</td></tr><tr><td colspan="4">Refer to product datasheets for details on hazards of using the reagents described here.</td></tr></tbody></table>

analyzing the effects of stromal cells on the recruitment of leukocytes from flow

基质细胞调节通过与邻近血管内皮细胞的串扰炎症时白细胞流传的招聘。在这里，我们介绍两个体外 "血管"模型研究了发炎的内皮细胞流动循环中性粒细胞的招募。这些模型的一个主要优点是分析中的白细胞粘附级联的每个步骤，以便，如将发生在体内的能力。我们还描述了这两种模式如何可以适于研究的基质细胞的作用，在这种情况下，间充质干细胞（MSC），在调节白细胞募集。 初级内皮细胞单独或一起进行培养与人类的MSC在Ibidi microslides或在Transwell滤膜24小时的相对侧直接接触。培养物刺激肿瘤坏死因子（TNFα）4小时，掺入基于流的粘附试验。嗜中性粒细胞的丸灌注在内皮4分钟。嗜中性粒细胞流入和它们与内皮相互作用的捕获是可视化通过相衬显微镜。 在这两种模式，细胞因子刺激增加流动中性粒细胞内皮招聘呈剂量依赖性。招募嗜中性粒细胞的行为的分析表明剂量依赖性减少轧制和通过内皮的剂量依赖性增加轮回。在共培养，MSC抑制中性粒细胞粘附到TNFα刺激的内皮细胞。 我们基于流的粘连模型模拟白细胞招聘的初始阶段的循环。除了白细胞，它们可以被用来检查其它类型的细胞，如治疗给药的MSC或循环肿瘤细胞的募集。我们的多层共培养模型已经表明，MSC与内皮通信以修改其响应于促炎细胞因子，alteri纳克中性粒细胞的招募。采用这种模式需要进一步研究，充分了解来自不同组织和条件（炎症性疾病或癌症）如何基质细胞在炎症过程中影响白细胞的招募。

最初，我们分析了在使用Ibidi的MicroSlide模型（ 第7 - 9）从流嗜中性粒细胞的募集刺激EC与TNFα的影响。在不存在TNFα，如果小任何中性白细胞粘附到内皮细胞单层（ 图2A）的。这是意料之中的，因为未处理的/休息EC不表达必需的粘附分子（选择素）或趋化因子，以支持结合25,26。与此相反，细胞因子的刺激显著增加中性粒细胞粘附到内皮细胞中以剂量依赖的方式（ 图2A）。密合性，通常保持稳定的测定过程。白细胞对内皮未处理的结合表明，无论是EC被激活（ 即，混有在培养过程中，LPS）和/或嗜中性粒细胞中分离的过程被激活。事实上，内毒素已被证明能增加E-选择的表达，ICAM-1和VCAM-1 月25日至27日 EC的表面，让他们结合嗜中性粒细胞上。 当分析招募嗜中性粒细胞的行为，我们通常观察到的剂量依赖性降低嗜中性粒细胞轧制（ 图2B）与嗜中性粒细胞通过内皮细胞单层（ 图2C）迁移的百分比伴随剂量依赖性增加的百分比。在较低剂量的TNFα刺激（1U / ml）的嗜中性粒细胞的比例较大出现相亮，指示它们所连接的内皮（ 图2B）的顶表面上。与此相反，在10和100单位/毫升（高剂量）的招募嗜中性粒细胞的约40％出现相黑暗中在2分钟表明这些细胞已通过内皮单层迁移并且是内皮（ 图2C）的下方。嗜中性粒细胞能够通过EC迁移中的1 - 2分钟，用轮回达到米aximal水平〜10分钟后灌注28。在这里，我们通过9分钟后的灌注（ 图2C）观察到的增加嗜中性粒细胞轮回从40％在2分钟至60％。我们观察到在滚动（〜3微米/秒）或迁移（〜10 - 12微米/分钟）的速度的TNFα浓度没有影响嗜中性粒细胞。 在过滤器基模型，TNFα刺激增加中性粒细胞粘附以类似于使用Ibidi的MicroSlide模型（ 图3A）观察的剂量依赖性。在行为方面，嗜中性粒细胞轧制是不受TNFα的剂量（ 图3B），而剂量依赖性增加百分比轮回观察（ 图3C）。在这一系列的实验中，我们观察到的时间对嗜中性粒细胞的轮回（ 图3C）无显著影响。 我们提供了有关如何生成两个不同的共培养构建体的方法中，每个这是设计来回答具体问题。在Ibidi的MicroSlide模型，乳油和MSC是在彼此直接接触的单个单层培养的。这种模式是用于检查治疗性注射MSC的效果进入血液，并随后将其融入EC单层有用。在基于过滤器的模型对比，乳油和MSC是在靠近过滤器的相对侧上培养的，但不一定是直接接触。这更类似于组织，与内皮细胞形成表示血管的单层，和MSC居住在内皮下室中。这使我们能够审视组织​​居民MSC对EC的细胞因子刺激的反应的影响。 根据我们的经验，我们观察到，当EC刺激与100 U / ml的TNFα最大的中性粒细胞招聘和内皮迁移发生。因此，我们已经用这种浓度检查的MSC共培养的效果从流动中性粒细胞内皮招聘。在这里，我们在共培养呈现数据BMMSC使用EC号的MicroSlide和过滤器为基础的模型然而，其他类型的MSC还可以检查如 WJMSC。在这两种模式BMMSC的存在相比，EC单独培养（ 图4A），中性粒细胞粘附显著降低到欧盟。共培养对招募嗜中性粒细胞的行为没有任何影响，用轧和轮回相似水平上观察到EC单独或与MSC（ 图4B和C）的培养。因此，MSC可以修改EC反应的细胞因子的刺激，从而抑制其从流支持中性粒细胞的招聘能力。 <img alt="图1" src="/files/ftp_upload/52480/52480fig1highres.jpg" /> 图1.建立的EC-MSC共培养，并使用基于流的粘附实验分析中性粒细胞募集。（A）中 </强&gt;显微（ⅰ）初级EC和（ⅱ）通道3 BMMSC的生长在组织培养瓶中。乳油和MSC（B）的显微照片上培养6孔Transwell滤膜插入。灌注系统（C）的示意图用于产生（一）牢牢地附着（FA）及（ii）transmigrated（TM）以下的中性粒细胞从流通招聘EC流动。（D）平行板式过滤器流室的示意图。（E）显微照片100 U / ml的TNFα刺激。 图片C和D是从图2和图3中的方法对分子生物学采取:T细胞贩运，2010年，第53-54页28与施普林格科学与商业媒体类型的许可， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52480/52480fig1large.jpg" target="_blank">请点击这里查看这是一个更大的版本数字。</a> 。使用Ibidi microslides图2.中性粒细胞募集，从流TNFα刺激的EC指令被刺激浓度的TNFα的增加（0 - 100U / ml）处理4小时。 A 4分钟丸粒细胞灌注在EC单层0.05帕。（A）中性粒细胞粘附在2分钟评估。方差分析表明TNFα治疗对中性粒细胞粘附，P &lt;0.01显著效果。嗜中性粒细胞的行为进行评估，在2和9分钟，并表示为贴壁细胞的百分比分别是（B）的轧制或（C）transmigrated。方差分析显示对中性粒细胞粘附，P &lt;0.001的行为TNFα治疗的显著效果。在C，ANOVA表现出显著的时间transmigrated中性粒细胞P &lt;0.01。数据是平均值±SEM从n = 3的实验。 * P &lt;0.05和**P &lt;0.01相对于未刺激的（0单位/毫升）乳油控制进行Dunnett试验后的## P &lt;0.01和### P &lt;0.001相对于1U / ml的EC在由邦弗朗尼后相同时间点试验。 <img alt="图3" src="/files/ftp_upload/52480/52480fig3highres.jpg" /> 从流动到TNFα刺激的EC使用滤波器为基础的检测图3.中性粒细胞募集乳油刺激与浓度的TNFα的增加（0 - 100U / ml）处理4小时。 A 4分钟丸粒细胞灌注在EC单层为0.1Pa。（A）中性粒细胞粘附在2分钟评估。方差分析表明TNFα治疗对中性粒细胞粘附，P &lt;0.001一个显著的效果。嗜中性粒细胞的行为进行评估，在2和9分钟，并表示为贴壁细胞的百分比分别是（B）的轧制或（C）transmigrated。在C，ANOVA笑结婚对中性粒细胞轮回，P &lt;0.05的时间和细胞因子治疗显著效果。数据是平均值±SEM从n = 3的实验。 ** P &lt;0.01和*** P &lt;0.001相对于未刺激的（0单位/毫升）乳油控制进行Dunnett试验后的。＃P &lt;0.05相比于1U / ml的EC在由邦弗朗尼交同一时间点-test。 <img alt="图4" src="/files/ftp_upload/52480/52480fig4highres.jpg" /> 图4.中性粒细胞募集，从流TNFα刺激的EC-BMMSC共培养物。BMMSC共培养与EC 24小时刺激之前用100U / ml的TNFα4小时。 A 4分钟丸粒细胞灌注在EC单层0.05霸为（A，C，E）microslides和0.1帕的（B，D，F）的过滤器。（AB）中性粒细胞粘附在2分钟评估。中性粒细胞的行为在2和9米评估并表示为贴壁细胞的百分比分别为:（CD）或滚动（EF）transmigrated。在C和D，方差分析显示培养条件对嗜中性粒细胞的滚动，P &lt;0.05显著效果。在E和 F，方差分析结果表明中性粒细胞轮回，P &lt;0.05的时间显著的效果。然而，没有显著差异由邦弗朗尼检测后，观察在个别处理之间轮回。数据是平均值±SEM从N = 5次实验。 * P &lt;0.05相比，EC单一配对t检验或邦费罗尼后的测试。 中性粒细胞的滚动速度的 EC对Transwell滤膜培养补充视频1.分析。刺激用100 U / ml的TNFα4小时。嗜中性粒细胞的丸灌注在EC为4分钟。一个10秒的领域有代表性的数字序列，采取2分钟后灌注。在从开始到10秒的序列的末端的单个轧制嗜中性粒细胞的位置的变化可被用于计算速度，在该嗜中性粒细胞是滚动。 的中性粒细胞迁移的速度补充视频2.分析。乳油培养在Transwell滤膜进行刺激以100U / ml的TNFα4小时。嗜中性粒细胞的丸灌注在EC 4分钟。单个5分钟字段代表数字化的序列，以跟踪transmigrated嗜中性粒细胞的移动。这可以被用来计算速度。

Watch this Scientific Journal Video about Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow at JoVE.com

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

发炎的内皮从流动招募白细胞的能力是由间充质基质细胞调节。我们描述两个体外结合原代人细胞，它可以用于从流动评估中性粒细胞募集和检查的作用是间充质基质细胞在调节这个过程中发挥的模型。

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

analyzing-effects-stromal-cells-on-recruitment-leukocytes-from

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.2K 次观看.  University of Birmingham. 发炎的内皮从流动招募白细胞的能力是由间充质基质细胞调节。我们描述两个体外结合原代人细胞，它可以用于从流动评估中性粒细胞募集和检查的作用是间充质基质细胞在调节这个过程中发挥的模型。 

视频: Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

科研

发育生物学

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

生物工程

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

免疫与感染

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

建模化疗耐药白血病<em&gt;体外</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

医学

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...