这项工作得到了欧洲血液学协会 2021 年研究高级资助、美国血液学会 2021 年全球研究奖以及欢迎信托基金和爱丁堡大学资助的内部战略支持基金 ISSF3 的支持。我们感谢流式细胞术设施的 Fiona Rossi 在流式细胞术分析方面的支持。出于开放获取的目的，作者已将知识共享署名 （CC BY） 许可应用于本次提交产生的任何作者接受的手稿版本。

hiPSC 来源的内皮细胞的分化和机械刺激

<ol>
	<li>Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+before+birth:+clues+from+gene+knockouts+and+mutations.">Death before birth: clues from gene knockouts and mutations.</a> Trends in Genetics. 11 (3), 87-93 (1995).</li><li>Ji, R. P., 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=Onset+of+cardiac+function+during+early+mouse+embryogenesis+coincides+with+entry+of+primitive+erythroblasts+into+the+embryo+proper.">Onset of cardiac function during early mouse embryogenesis coincides with entry of primitive erythroblasts into the embryo proper.</a> Circulation Research. 92 (2), 133-135 (2003).</li><li>Peacock, H. M., Daems, M., Jones, E. A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+control+of+endothelial+cell+fates+in+development.">Hemodynamic control of endothelial cell fates in development.</a> Cardiac and Vascular Biology. 8, 127-166 (2021).</li><li>Chong, D. C., Koo, Y., Xu, K., Fu, S., Cleaver, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+arteriovenous+fate+acquisition+during+mammalian+vasculogenesis.">Stepwise arteriovenous fate acquisition during mammalian vasculogenesis.</a> Developmental Dynamics. 240 (9), 2153-2165 (2011).</li><li>Jaffredo, T., Gautier, R., Eichmann, A., Dieterlen-Li&#232;vre, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraaortic+hemopoietic+cells+are+derived+from+endothelial+cells+during+ontogeny.">Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.</a> Development. 125 (22), 4575-4583 (1998).</li><li>Zovein, A. 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=Fate+Tracing+reveals+the+endothelial+origin+of+hematopoietic+stem+cells.">Fate Tracing reveals the endothelial origin of hematopoietic stem cells.</a> Cell Stem Cell. 3 (6), 625-636 (2008).</li><li>Bertrand, J. Y., 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=Haematopoietic+stem+cells+derive+directly+from+aortic+endothelium+during+development.">Haematopoietic stem cells derive directly from aortic endothelium during development.</a> Nature. 464 (7285), 108-111 (2010).</li><li>Boisset, J. 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=In+vivo+imaging+of+haematopoietic+cells+emerging+from+the+mouse+aortic+endothelium.">In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.</a> Nature. 464 (7285), 116-120 (2010).</li><li>Adamo, L., 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=Biomechanical+forces+promote+embryonic+haematopoiesis.">Biomechanical forces promote embryonic haematopoiesis.</a> Nature. 459 (7250), 1131-1135 (2009).</li><li>Diaz, M. F., 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=Biomechanical+forces+promote+blood+development+through+prostaglandin+E2+and+the+cAMP-PKA+signaling+axis.">Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.</a> Journal of Experimental Medicine. 212 (5), 665-680 (2015).</li><li>North, T. 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=Hematopoietic+stem+cell+development+is+dependent+on+blood+flow.">Hematopoietic stem cell development is dependent on blood flow.</a> Cell. 137 (4), 736-748 (2009).</li><li>Lundin, V., 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=YAP+regulates+hematopoietic+stem+cell+formation+in+response+to+the+biomechanical+forces+of+blood+flow.">YAP regulates hematopoietic stem cell formation in response to the biomechanical forces of blood flow.</a> Developmental Cell. 52 (4), 446.e5-460.e5 (2020).</li><li>Li, J., 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=Mimicry+of+embryonic+circulation+enhances+the+hoxa+hemogenic+niche+and+human+blood+development.">Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development.</a> Cell Reports. 40 (11), 111339 (2022).</li><li>Azzoni, 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=The+onset+of+circulation+triggers+a+metabolic+switch+required+for+endothelial+to+hematopoietic+transition.">The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition.</a> Cell Reports. 37 (11), 110103 (2021).</li><li>Li, Y. S. J., Haga, J. H., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+effects+of+shear+stress+on+vascular+endothelial+cells.">Molecular basis of the effects of shear stress on vascular endothelial cells.</a> Journal of Biomechanics. 38 (10), 1949-1971 (2005).</li><li>Batsivari, A., 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=Understanding+hematopoietic+stem+cell+development+through+functional+correlation+of+their+proliferative+status+with+the+intra-aortic+cluster+architecture.">Understanding hematopoietic stem cell development through functional correlation of their proliferative status with the intra-aortic cluster architecture.</a> Stem Cell Reports. 8 (6), 1549-1562 (2017).</li><li>Canu, 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=Analysis+of+endothelial-to-haematopoietic+transition+at+the+single+cell+level+identifies+cell+cycle+regulation+as+a+driver+of+differentiation.">Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation.</a> Genome Biology. 21 (1), 157 (2020).</li><li>Luo, W., 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=Arterialization+requires+the+timely+suppression+of+cell+growth.">Arterialization requires the timely suppression of cell growth.</a> Nature. 589 (7842), 437-441 (2020).</li><li>Yang, C. -. T., 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=Activation+of+KLF1+enhances+the+differentiation+and+maturation+of+red+blood+cells+from+human+pluripotent+stem+cells.">Activation of KLF1 enhances the differentiation and maturation of red blood cells from human pluripotent stem cells.</a> Stem Cells. 35 (4), 886-897 (2017).</li><li>Lopez-Yrigoyen, 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=A+human+iPSC+line+capable+of+differentiating+into+functional+macrophages+expressing+ZsGreen:+A+tool+for+the+study+and+in+vivo+tracking+of+therapeutic+cells.">A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: A tool for the study and in vivo tracking of therapeutic cells.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 373 (1750), 20170219 (2018).</li><li>Lopez-Yrigoyen, 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=Production+and+characterization+of+human+macrophages+from+pluripotent+stem+cells.">Production and characterization of human macrophages from pluripotent stem cells.</a> Journal of Visualized Experiments. 2020 (158), (2020).</li><li>Fidanza, A., 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=Single+cell+analyses+and+machine+learning+define+hematopoietic+progenitor+and+HSC-like+cells+derived+from+human+PSCs.">Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs.</a> Blood. 136 (25), 2893-2904 (2020).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>North, T. 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=Hematopoietic+stem+cell+development+is+dependent+on+blood+flow.">Hematopoietic stem cell development is dependent on blood flow.</a> Cell. 137 (4), 736-748 (2009).</li><li>Nguyen, J., Lin, Y. Y., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+generation+of+endothelial+differentiation:+Tissue-specific+ECs.">The next generation of endothelial differentiation: Tissue-specific ECs.</a> Cell Stem Cell. 28 (7), 1188-1204 (2021).</li><li>Petazzi, P., 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=Arterial+cells+support+the+development+of+human+hematopoietic+progenitors+in+vitro+via+secretion+of+IGFBP2.">Arterial cells support the development of human hematopoietic progenitors in vitro via secretion of IGFBP2.</a> bioRxiv. , (2022).</li><li>Crosse, E. I., 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=Multi-layered+spatial+transcriptomics+identify+secretory+factors+promoting+human+hematopoietic+stem+cell+development.">Multi-layered spatial transcriptomics identify secretory factors promoting human hematopoietic stem cell development.</a> Cell Stem Cell. 27 (5), 822 (2020).</li><li>Calvanese, V., 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=Mapping+human+haematopoietic+stem+cells+from+haemogenic+endothelium+to+birth.">Mapping human haematopoietic stem cells from haemogenic endothelium to birth.</a> Nature. 604 (7906), 534-540 (2022).</li><li>Zeng, Y., 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=Tracing+the+first+hematopoietic+stem+cell+generation+in+human+embryo+by+single-cell+RNA+sequencing.">Tracing the first hematopoietic stem cell generation in human embryo by single-cell RNA sequencing.</a> Cell Research. 29 (11), 881-894 (2019).</li><li>Hwa, J. J., 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=Abnormal+arterial-venous+fusions+and+fate+specification+in+mouse+embryos+lacking+blood+flow.">Abnormal arterial-venous fusions and fate specification in mouse embryos lacking blood flow.</a> Scientific Reports. 7 (1), 11965 (2017).</li></ol>

作者没有要披露的利益冲突。

我们在这里描述的方案允许从人多能干细胞中产生机械敏感的内皮细胞，并研究它们对由受控剪切应力介导的机械刺激的反应。该方案完全基于细胞因子，与GMP试剂完全兼容，可潜在转化为细胞治疗的细胞生产。
hiPSCs的推导为科学家提供了胚胎发育早期阶段的仪器模型，使研究难以在体内研究的过程成为可能24。事实上，可用于研究的人类胚胎组织是从缺乏循环的胚胎中收集的，这可能对机械线索控制的分子特征产生重大影响。这里描述的方法能够对细胞对剪切应力的反应进行实时成像和实时研究。hiPSCs与流体的结合提供了一种研究模型，该模型克服了循环开始重塑和控制心血管和血液系统建立时发育中的胎儿组织的有限可用性和不可接近性3,9,10,25。
该方案的一个局限性是，从该方案衍生的内皮细胞可能无法反映发育组织中存在的不同内皮细胞的各种身份。为了克服这一局限性，在流体刺激之前的分化过程中可能需要特定的细胞因子组合，以获得所需的身份或组织特异性表型26。在分离步骤中，可以使用更精细的免疫表型获得内皮亚群的分离。该方案仅基于CD34的表达分离内皮细胞，从而允许柱分离而不是荧光激活细胞分选（FACS）;这减少了细胞死亡和污染风险。此外，该协议专门用于研究层流介导的剪切应力的作用。必须采用替代流体方法来研究其他机械线索的影响，例如拉伸或压缩，或其他类型的流动，例如扰动或扰动流动。
我们之前已经证明，iPSC 衍生的内皮细胞模仿异质动静脉细胞身份27，类似于在胎儿背主动脉28、29、30 中观察到的身份。这在已知由血液循环控制的血管发育和细胞规格方面尤为重要。不同模型的研究表明，缺乏循环会导致动静脉规格改变11,14,31。将机械线索与细胞规格联系起来的机制仍然未知，这里描述的管道允许进行无法在体内测试的精细功能研究。
该管道描述了使用市售流体通道生产和刺激源自 hiPSC 的内皮细胞，避免了像广泛使用的聚二甲基硅氧烷 （PDMS） 装置那样铸造装置的需要12。此外，PDMS芯片的使用使得刺激细胞的收集特别具有挑战性，而使用该协议，可以很容易地从通道中取出细胞。这显着提高了分析能力，允许后续分析，例如蛋白质组学和转录组学分析、流式细胞术和功能测定，这些可能需要进一步的培养或 体内 测定。

在胚胎发育过程中，心脏是第一个建立功能的器官1，从心内膜管形成的最早阶段2 开始就可检测到收缩。循环，以及由血管内血液流动介导的机械线索，控制着早期发育的许多关键方面。在胎儿循环建立之前，脉管系统被组织成一个初级毛细血管丛;心脏功能正常后，该神经丛重组为静脉和动脉脉管系统3。机械线索在动静脉规范中的作用反映在血流开始前动脉和静脉标志物的泛内皮表达4.
血流动力学不仅控制着脉管系统本身的发育，而且在控制血细胞形成中起着基础性作用。造血干细胞和祖细胞 （HSPC） 从称为造血内皮细胞5、6、7、8 的特殊内皮细胞中出现，仅在发育早期存在于胚胎的不同解剖区域。心脏缺陷模型与体外模型一起证明，机械线索指示并增加血液源性内皮细胞 HSPC 的产生 9,10,11,12,13,14。
不同类型的血流动力学已被证明可以差异控制细胞周期15，已知在血源性内皮 16,17 和动脉细胞规范18 中都很重要。总而言之，机械线索是发育过程中细胞身份和功能的关键决定因素。新型体外流体装置使我们能够克服在体内人类血液发育过程中研究发育机械生物学的局限性。
本手稿中方案的总体目标是逐步描述实验管道，以研究剪切应力对 体外 来源于人诱导多能干细胞（hiPSC）的人内皮细胞的影响。该方案包含有关将hiPSC分化为内皮细胞及其随后接种到用于刺激方案的流体芯片中的详细说明。使用这种方法，可以通过分析不同的 体外来源的内皮细胞响应流动的方向来测试它们感知剪切应力的能力。这将使其他实验室能够解决有关剪切应力的响应及其对不同内皮细胞身份的功能影响的问题。

<table><tbody><tr><td>0.6 Luer uncoated slide</td><td>ibidi</td><td>IB-80186</td><td></td></tr><tr><td>25% BSA</td><td>Life Technologies</td><td>A10008-01</td><td></td></tr><tr><td>6-well plates</td><td>Greiner Bio-one</td><td>657160</td><td></td></tr><tr><td>Accutase</td><td>Life Technologies</td><td>A1110501</td><td>Cited as Dissociation reagent</td></tr><tr><td>Ascorbic acid</td><td>Merck</td><td>A4544-100G</td><td></td></tr><tr><td>Aspiration pipette</td><td>Sardtedt</td><td>86.1252.011</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504044</td><td>Cited as Neuronal cell culture supplement (50x)</td></tr><tr><td>BD FACS DIVA</td><td>BD Biosciences</td><td>Version 8.0.1</td><td>Cited as flow cytometry software</td></tr><tr><td>BD LSR Fortessa 5 Laser</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>bFGF</td><td>Life Technologies</td><td>PHG0021</td><td></td></tr><tr><td>CD34 Microbead kit</td><td>Miltenyil Biotec</td><td>30-046-702</td><td></td></tr><tr><td>CD34 PE clone 4H11</td><td>Invitrogen</td><td>12-0349-42</td><td></td></tr><tr><td>CD34 PerCP-eFluor 710 clone 4H11</td><td>Invitrogen</td><td>44-0349-42</td><td></td></tr><tr><td>Cellstar cell-repellent surface 6-well plates</td><td>Greiner Bio-one</td><td>657970</td><td>Cited as cell-repellent plate</td></tr><tr><td>CHIR99021</td><td>Cayman Chemicals&amp;nbsp;</td><td>13122-1mg-CAY</td><td></td></tr><tr><td>Cryostor CS10&amp;nbsp; cell cryopreservation</td><td>Merck</td><td>C2874-100ML</td><td>Cited as Cryopreservation solution</td></tr><tr><td>Dimethyl Sulfoxide</td><td>VWR</td><td>200-664-3</td><td>Cited as DMSO</td></tr><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>DPB Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080055</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14200075</td><td></td></tr><tr><td>EASY Strainer 40 &amp;mu;m</td><td>Greiner Bio-one</td><td>542040</td><td></td></tr><tr><td>EDTA</td><td>Life technologies</td><td>15575020</td><td></td></tr><tr><td>FcR Blocking Reagent</td><td>Miltenyil Biotec</td><td>130-059-901</td><td></td></tr><tr><td>Fiji</td><td></td><td>Version 1.53c</td><td></td></tr><tr><td>Flow Jo</td><td></td><td>Version 10.7.1</td><td>Cited as flow cytometry sanalysis oftware</td></tr><tr><td>FLT3L</td><td>Peprotech</td><td>300-19-10uG</td><td></td></tr><tr><td>Fluidic unit</td><td>ibidi</td><td>10903</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050038</td><td>Cited as L-glutamine supplement</td></tr><tr><td>Ham F-12&amp;nbsp;</td><td>Life Technologies</td><td>11765054</td><td></td></tr><tr><td>Holo-transferrin</td><td>Merk</td><td>T0665-500MG</td><td></td></tr><tr><td>Human Serum Albumin</td><td>Fujifilm UK LTD</td><td>9988</td><td></td></tr><tr><td>Ibidi Pump system</td><td>ibidi</td><td>10902</td><td>Cited as Pump system</td></tr><tr><td>IMDM</td><td>Life Technologies</td><td>12440053</td><td></td></tr><tr><td>Inverted microscope</td><td>ioLight/Thisle Scientific</td><td>IOL-IO-INVERT</td><td>Cited as inverted in-incubator microscope</td></tr><tr><td>Lyophilised BSA&amp;nbsp;</td><td>Merck</td><td>A2153-100G</td><td></td></tr><tr><td>MiniMACS Separator</td><td>Miltenyil Biotec</td><td>130-042-102</td><td>Cited as Magnetic separator</td></tr><tr><td>MS Columns</td><td>Miltenyil Biotec</td><td>130-042-201</td><td>Cited as Magnetic column</td></tr><tr><td>MTG</td><td>Merck</td><td>M6145-25ML</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td></td></tr><tr><td>Notebook for pump system</td><td>ibidi</td><td>10908</td><td></td></tr><tr><td>Paraformaldehyde 37-41%</td><td>Fisher Chemicals</td><td>F/1501/PB15</td><td></td></tr><tr><td>Pastette</td><td>Greiner Bio-one</td><td>612398</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Perfusion Set YELLOW/GREEN: 50 cm, ID 1.6 mm, 10 mL reservoirs</td><td>Ibidi</td><td>IB-10964</td><td>Cited as Perfusion set</td></tr><tr><td>Polystyrene Round Bottom Tubes</td><td>Falcon</td><td>352008</td><td>Cited as Flow cytometry tubes</td></tr><tr><td>Prism 9</td><td></td><td>Verison 9.4.0</td><td></td></tr><tr><td>Pump control software</td><td>ibidi</td><td>version 1.6.1</td><td>Cited as Pump software</td></tr><tr><td>ReLeSR</td><td>Stem cell tecchonologies</td><td>5872</td><td>Cited as Detaching solution</td></tr><tr><td>rhBMP4</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>rhEPO</td><td>R&amp;amp;D</td><td>287-TC-500</td><td></td></tr><tr><td>rhIGF1</td><td>Peprotech</td><td>100-11-100uG</td><td></td></tr><tr><td>rhIL11</td><td>Peprotech</td><td>200-11-10uG</td><td></td></tr><tr><td>rhIL3</td><td>Peprotech</td><td>200-03-10uG</td><td></td></tr><tr><td>rhIL6</td><td>R&amp;amp;D</td><td>206-IL-010</td><td></td></tr><tr><td>rhLaminin-521</td><td>Life technologies</td><td>A29248</td><td>Cited as Laminin</td></tr><tr><td>rhSCF</td><td>Life Technologies</td><td>PHC2111</td><td></td></tr><tr><td>rhTPO</td><td>R&amp;amp;D</td><td>288-TPN-025</td><td></td></tr><tr><td>rhVEGF</td><td>R&amp;amp;D</td><td>293-VE-010</td><td></td></tr><tr><td>RLT Lysis Buffer</td><td>Qiagen</td><td>79216</td><td></td></tr><tr><td>Serial Connector for &amp;micro;-Slides: Sterile, Sterile</td><td>ibidi</td><td>IB-10830</td><td></td></tr><tr><td>StemPro-CD34 SFM media</td><td>Life Technologies</td><td>10639011</td><td>Cited as Serum-Free media for CD34+ cells (SFM-34)</td></tr><tr><td>StemPro-CD34 Nutrient Supplement&amp;nbsp;</td><td>Life Technologies</td><td>10641-025</td><td>Cited as 34 nutrient supplement</td></tr><tr><td>StemPro hESC SFM</td><td>Life Technologies</td><td>A1000701</td><td>Cited as Culture media&amp;nbsp;</td></tr><tr><td>StemPro supplement</td><td>Life Technologies</td><td>A10006-01</td><td></td></tr><tr><td>Vitronectin (VTN-N) recombinant human protein, truncated</td><td>Invitrogen</td><td>A31804</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td>Cited as iRock</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr></tbody></table>

in vitro model of fetal human vessel on-chip to study developmental mechanobiology

心脏是发育过程中第一个功能建立的器官，因此在妊娠早期就启动了血液循环。除了运输氧气和营养物质以确保胎儿生长外，胎儿循环还通过机械线索控制内皮层内发生的许多关键发育事件。生物力学信号诱导血管结构变化，建立动静脉规范，控制造血干细胞的发育。发育中的组织无法接近限制了对循环在人类早期发育中的作用的理解;因此， 体外 模型是研究船舶机械生物学的关键工具。本文描述了一种将内皮细胞与人诱导的多能干细胞区分开来的方案，并将其随后接种到流体装置中以研究它们对机械线索的反应。这种方法允许在机械刺激下长期培养内皮细胞，然后取回内皮细胞以进行表型和功能表征。这里建立的 体外 模型将有助于阐明细胞内分子机制，这些机制转导由机械线索介导的信号传导，最终协调人类胎儿生命中的血管发育。

我们在这里描述了一种用于分化和机械刺激源自hiPSC的内皮细胞的方案，该方案允许研究它们对机械线索的反应（图1）。该方案导致功能机械敏感内皮细胞的产生。我们在这里提供具有代表性的结果并描述预期的表型，以评估细胞在分化过程中对细胞因子刺激的反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65492/65492fig01v2.jpg"> 图 1：分化和机械刺激方案的示意图。 分化方案示意图，显示了不同细胞因子混合物的时间、CD34+ 细胞分离、流控芯片接种和机械刺激细胞的最终分析。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig01largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
hiPSCs培养 重要的是从在自我更新条件下正确生长的hiPSC开始方案。文化质量的一个很好的指标是它们的生长速度。解冻后，细胞可能需要 2-3 周才能达到正确的生长阶段，以确保良好的分化。当细胞可以每周以 1：6 的比例传代两次，达到几乎完全汇合时，这是它们在需要传代的同一天准备分化的时间。
hiPSCs分化为内皮细胞 分化的第一步，包括胚状体（EBs）的形成，是细胞系依赖性的，可能需要对使用的特定细胞系进行一些优化。方案步骤1.3.2.2-1.3.2.4中描述的解离可以通过减少或延长与解离试剂的孵育以及随后与巴斯德移液管的解离来修饰。此外，除了使用切割工具或P100移液器吸头对菌落进行物理解离外，还可以使用其他解离试剂进行此步骤。质量好的EB在分化的第2天显示出明确的边缘，当使用显微镜观察时，它看起来清晰明亮;较暗的区域可能表明EB内的细胞死亡（图2）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65492/65492fig2v3.jpg"> 图2：胚状体形态 。 （A） 第 2 天胚状体显示轮廓分明的外缘和一致的大小。（B） 第 2 天，质量较差的胚状体显示广泛的细胞死亡，导致结构分解。比例尺 = 500 μm。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
在第 2 天，向 EB 中添加 CHIR99021 会抑制 GSK-3 蛋白，从而激活 Wnt 通路。不同的细胞系对CHIR处理有不同的反应，这应该通过使用不同的浓度量化第8天获得的CD34 + 细胞的数量来测试（图3）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65492/65492fig3v3.jpg"> 图 3：不同 CHIR 处理下的内皮细胞分化。 在第 2 天以 （A） 3 μM、（B） 5 μM 和 （C） 7 μM 进行 CHIR 处理后，通过流式细胞术在 CD34 膜表达分化第 8 天通过流式细胞术定量内皮细胞承诺。 使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
CD34+ 细胞分离 在洗脱色谱柱后，验证使用磁珠富集的CD34+是否至少提供80%的CD34+非常重要。为确保足够的纯度，可以通过流式细胞术分析从磁性分离中获得的细胞等分试样，确保使用与用于磁富集的抗体克隆不同的抗体克隆。在这里，使用4H11克隆，富集后纯度达到~85%（图4）。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65492/65492fig4v3.jpg"> 图4：磁分选富集前后CD34的膜表达。 对第 8 天解离的胚状体（灰色）和磁富集后的细胞（绿色）进行 CD34 表达染色，并通过流式细胞术进行分析，显示分选后成功富集。使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
将细胞接种到流体通道中 在流体通道中接种细胞时，跟踪内皮细胞的粘附和增殖至关重要。接种后，细胞需要~5小时才能完全粘附在通道上（图5A）。在此阶段，还可以测试替代涂层解决方案以提高附着力。为了确认被测细胞对机械敏感，因此能够对机械刺激做出反应，可以随时间推移测试细胞取向。刺激前的细胞显示随机取向（图5A和图5C），并且它们平行于流动方向重新定向（图5B，C）。这里描述的方案允许从通道中收集细胞以进行下游分析，例如流式细胞术，以研究其膜免疫表型，提供受刺激细胞的内皮特性（图5D，E）。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65492/65492fig5v3.jpg"> 图 5：hiPSCs 衍生的内皮细胞的机械反应性 。 （A） 接种后 48 小时分离的 CD34+ 细胞汇合层。（B） 动态培养 3 天的内皮细胞重新定向层。（C） 动态培养 5 天后内皮细胞的取向分析。（D） 流动培养 5 天的细胞的 CD34 表达谱。（E） 从流体通道中检索到的细胞群中 CD34+ 细胞的百分比。使用倒置培养箱显微镜拍摄图像;使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。比例尺 = 100 μm （A，B）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>库存集中度</td>
			<td>添加的音量</td>
			<td>最终浓度</td>
		</tr><tr><td>Iscove 改良 Dulbecco 培养基 （IMDM）</td>
			<td>-</td>
			<td>333毫升</td>
			<td>-</td>
		</tr><tr><td>Ham's F-12 营养混合物 （F-12）</td>
			<td>-</td>
			<td>167毫升</td>
			<td>-</td>
		</tr><tr><td>N-2 补充剂 （100x）</td>
			<td>100 倍</td>
			<td>5毫升</td>
			<td>1 倍</td>
		</tr><tr><td>B-27 补充剂 （50x）</td>
			<td>50 倍</td>
			<td>10毫升</td>
			<td>1 倍</td>
		</tr><tr><td>抗坏血酸</td>
			<td>10毫克/毫升</td>
			<td>1.25毫升</td>
			<td>25微克/毫升</td>
		</tr><tr><td>α-单硫代甘油 （MTG）</td>
			<td>11.5 米</td>
			<td>19.5微升</td>
			<td>448.5微米</td>
		</tr><tr><td>人血清白蛋白</td>
			<td>100毫克/毫升</td>
			<td>2.5毫升</td>
			<td>0.5毫克/毫升</td>
		</tr><tr><td>全转铁蛋白</td>
			<td>100毫克/毫升</td>
			<td>0.75毫升</td>
			<td>150微克/毫升</td>
		</tr></tbody></table>表 1：500 mL 无血清分化 （SFD） 培养基的组成和配方。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>分化天数</td>
			<td>细胞因子混合物</td>
			<td>细胞因子</td>
			<td>最终浓度</td>
		</tr><tr><td>第 0 - 2 天</td>
			<td>混合 1</td>
			<td>BMP4型</td>
			<td>20纳克/毫升</td>
		</tr><tr><td>第2天</td>
			<td>混合 2</td>
			<td>CHIR99021</td>
			<td>7微米</td>
		</tr><tr><td>从第 3 天开始</td>
			<td rowspan="2">混合 3 和 4</td>
			<td>血管内生长因子（VEGF）</td>
			<td>15纳克/毫升</td>
		</tr><tr><td></td>
			<td>bFGF系列</td>
			<td>5纳克/毫升</td>
		</tr><tr><td>从第 6 天开始</td>
			<td rowspan="8">混合 4</td>
			<td>IL6型</td>
			<td>10纳克/毫升</td>
		</tr><tr><td></td>
			<td>FLT3L型</td>
			<td>10纳克/毫升</td>
		</tr><tr><td></td>
			<td>胰岛素样生长因子1</td>
			<td>25纳克/毫升</td>
		</tr><tr><td></td>
			<td>IL11型</td>
			<td>5纳克/毫升</td>
		</tr><tr><td></td>
			<td>云函数</td>
			<td>50纳克/毫升</td>
		</tr><tr><td></td>
			<td>欧洲专利局</td>
			<td>3 U/毫升</td>
		</tr><tr><td></td>
			<td>热塑性聚氨酯（TP</td>
			<td>30纳克/毫升</td>
		</tr><tr><td></td>
			<td>IL3型</td>
			<td>30纳克/毫升</td>
		</tr></tbody></table>表 2：用于内皮细胞分化的细胞因子混合物、将它们添加到 SFD 培养基中的天数和最终浓度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>剪切应力 （dyn/cm2）</td>
			<td>时间 （h）</td>
		</tr><tr><td>0.5</td>
			<td>1</td>
		</tr><tr><td>1</td>
			<td>1</td>
		</tr><tr><td>1.5</td>
			<td>1</td>
		</tr><tr><td>2</td>
			<td>1</td>
		</tr><tr><td>2.5</td>
			<td>1</td>
		</tr><tr><td>3</td>
			<td>1</td>
		</tr><tr><td>3.5</td>
			<td>1</td>
		</tr><tr><td>4</td>
			<td>1</td>
		</tr><tr><td>4.5</td>
			<td>1</td>
		</tr><tr><td>5</td>
			<td>直到实验结束</td>
		</tr></tbody></table>表 3：动态文化的剪切应力值及其应用时间。
补充图S1：用于该协议的芯片和管路的几何形状和尺寸。<a href="https://www.jove.com/files/ftp_upload/65492/SF1.pdf" target="_blank">请点击这里下载此文件。</a>
补充图 S2：控制气泵的软件的分步指南，并附有每个步骤的说明。<a href="https://www.jove.com/files/ftp_upload/65492/65492_Supp%20Figure%20S2%20FINAL.pdf" target="_blank">请点击这里下载此文件。</a>
补充图S3：使用FIJI进行取向分析的指南，显示了细胞形状的绘制、椭圆拟合和最终测量。<a href="https://www.jove.com/files/ftp_upload/65492/65492_SF3.pdf" target="_blank">请点击这里下载此文件。</a>
补充表S1：分化方案中使用的细胞因子的单位大小、重悬体积和储备液浓度。<a href="https://www.jove.com/files/ftp_upload/65492/Ventura%20et%20al,%202023%20-%20Supplementary%20Table%201.xlsx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation at JoVE.com

作者聚焦：研究在流体介导的机械刺激下培养的 hiPSC 衍生的内皮细胞 

这里描述的是将内皮细胞与人多能干细胞区分开来的简单工作流程，然后是其机械刺激的详细方案。这允许研究内皮细胞的发育机制生物学。该方法与机械刺激后从培养芯片收集的活细胞的下游测定兼容。

体外研究 胎儿人体血管芯片模型研究发育机械生物学

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

我们的研究探讨了机械线索如何在发育过程中指导内皮细胞的命运，为此我们将人类诱导的多能干细胞分化与流体介导的机械刺激相结合。内皮细胞不断暴露在血液运动中，因此在静态培养中会遗漏重要信号，这使得使用传统培养系统研究内皮细胞生物学成为主要挑战。事实上，机械线索激活了许多关键的信号通路，这些通路在静态条件下培养的细胞中可能被忽视。我们的方案提供了关于如何分化和机械刺激诱导多能干细胞衍生的内皮细胞的简单、分步指南。这将有助于许多实验室采用热培养，以轻松解决有关内皮细胞在流动中行为的问题。

in-vitro-model-fetal-human-vessel-on-chip-to-study-developmental

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

1.5K 次观看.  University of Edinburgh. 我们的研究探讨了机械线索如何在发育过程中指导内皮细胞的命运，为此我们将人类诱导的多能干细胞分化与流体介导的机械刺激相结合。内皮细胞不断暴露在血液运动中，因此在静态培养中会遗漏重要信号，这使得使用传统培养系统研究内皮细胞生物学成为主要挑战。事实上，机械线索激活了许多关键的信号通路，这些通路在静态条件下培养的细胞中可能被忽视。我?...

视频: 作者聚焦：研究在流体介导的机械刺激下培养的 hiPSC 衍生的内皮细胞 

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

科研

生物工程

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

发育生物学

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

研究组织刚性和剪切应力对内皮细胞的影响

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

作者聚焦：整合基质刚度和流动条件以研究血管力学生物学中的内皮细胞反应

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

整合基质刚度和流动性的体外模型以研究内皮细胞反应

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

体外研究胎儿人体血管芯片模型研究发育机械生物学

副本

摘要

探索更多视频

此视频中的章节

发展多深的圆形横截面微内皮化的一个芯片的程序

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

缩微和3D微血管大会

微流控装置中组织模型的 Perfusable 血管网

模仿神经血管化初始事件的微流体模型

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

整合基质刚度和流动性的体外模型以研究内皮细胞反应

整合基质刚度和流动性的体外模型以研究内皮细胞反应

整合基质刚度和流动性的体外模型以研究内皮细胞反应

体外研究 胎儿人体血管芯片模型研究发育机械生物学

副本

摘要

探索更多视频

此视频中的章节

发展多深的圆形横截面微内皮化的一个芯片的程序

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

缩微和3D微血管大会

微流控装置中组织模型的 Perfusable 血管网

模仿神经血管化初始事件的微流体模型

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

整合基质刚度和流动性的体外模型以研究内皮细胞反应

整合基质刚度和流动性的体外模型以研究内皮细胞反应

整合基质刚度和流动性的体外模型以研究内皮细胞反应

体外研究胎儿人体血管芯片模型研究发育机械生物学