Name: isogenic kidney glomerulus chip engineered from human induced pluripotent stem cells
Uploaded: 2022-11-04T13:15:00
Description: Watch this Scientific Journal Video about Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells at JoVE.com

这项工作得到了杜克大学普拉特工程学院、杜克大学医学系肾脏病学系、怀特黑德生物医学研究奖学金和S. Musah基因泰克研究奖的支持。Y. Roye是杜克大学-阿尔弗雷德·P·斯隆基金会奖学金和杜克大学生物医学工程系William M. “Monty” Reichert研究生奖学金的获得者。DU11（杜克大学克隆#11）iPS细胞系在杜克大学iPSC核心设施中产生，并由杜克大学的Bursac实验室提供给我们。作者感谢N. Abutaleb，J. Holmes，R. Bhattacharya和Y. Zhou的技术援助和有益的讨论。作者还要感谢Musah实验室的成员对手稿的有益评论。作者感谢Segura实验室赠送的Acuri C6流式细胞仪。

1. 制备基底膜基质溶液和涂层基材
<ol><li>解冻基底膜基质1在4°C冰上过夜。 根据制造商建议的稀释比例等分。使用 50 mL 锥形管和移液器，将适量的基底膜基质 1 与 25 mL 冷 DMEM/F12 充分混合，直至完全解冻并溶解。<ol><li>要溶解冷冻等分试样，请从 25 mL 冷 DMEM/F12 中取出 ~200 μL，并将其转移到冷冻等分试样管中。上下移液，直到基质彻底解冻并溶解。将基底膜基质 1 的全部管内容物转?...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+induced+pluripotent+stem+cells+into+mature+kidney+podocytes+and+establishment+of+a+Glomerulus+Chip.">Directed differentiation of human induced pluripotent stem cells into mature kidney podocytes and establishment of a Glomerulus Chip.</a> Nature Protocols. 13 (7), 1662-1685 (2018).</li><li>Burt, M., Bhattachaya, R., Okafor, A. E., Musah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+differentiation+of+mature+kidney+podocytes+from+human+induced+pluripotent+stem+cells+under+chemically+defined+conditions.">Guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions.</a> Journal of Visualized Experiments. (161), e61299 (2020).</li><li>Roye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personalized+glomerulus+chip+engineered+from+stem+cell-derived+epithelium+and+vascular+endothelium.">A personalized glomerulus chip engineered from stem cell-derived epithelium and vascular endothelium.</a> Micromachines. 12 (8), 967 (2021).</li><li>Roselli, S., 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=Podocin+localizes+in+the+kidney+to+the+slit+diaphragm+area.">Podocin localizes in the kidney to the slit diaphragm area.</a> The American Journal of Pathology. 160 (1), 131-139 (2002).</li><li>Ruotsalainen, 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=Nephrin+is+specifically+located+at+the+slit+diaphragm+of+glomerular+podocytes.">Nephrin is specifically located at the slit diaphragm of glomerular podocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (14), 7962-7967 (1999).</li><li>Privratsky, J. R., Newman, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PECAM-1:+regulator+of+endothelial+junctional+integrity.">PECAM-1: regulator of endothelial junctional integrity.</a> Cell and Tissue Research. 355 (3), 607-619 (2014).</li><li>Menon, M. C., Chuang, P. Y., He, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glomerular+filtration+barrier:+components+and+crosstalk.">The glomerular filtration barrier: components and crosstalk.</a> International Journal of Nephrology. 2012, 749010 (2012).</li><li>Abutaleb, N. O., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+characterization+of+human+iPSC-derived+vascular+endothelial+cells+under+physiological+shear+stress.">Differentiation and characterization of human iPSC-derived vascular endothelial cells under physiological shear stress.</a> STAR Protocols. 2 (2), 100394 (2021).</li><li>Pammolli, F., Magazzini, L., Riccaboni, 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+productivity+crisis+in+pharmaceutical+R&amp;D.">The productivity crisis in pharmaceutical R&amp;D.</a> Nature Reviews. Drug Discovery. 10 (6), 428-438 (2011).</li><li>Atchison, 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=iPSC-derived+endothelial+cells+affect+vascular+function+in+a+tissue-engineered+blood+vessel+model+of+Hutchinson-Gilford+progeria+syndrome.">iPSC-derived endothelial cells affect vascular function in a tissue-engineered blood vessel model of Hutchinson-Gilford progeria syndrome.</a> Stem Cell Reports. 14 (2), 325-337 (2020).</li></ol>

在本报告中，我们概述了从同基因人iPS细胞系中获得血管内皮和肾小球上皮（足细胞）的方案，并使用这些细胞来设计3D器官芯片系统，该系统模拟肾小球的结构，组织 - 组织界面和分子过滤功能。这种肾小球芯片配备了内皮和肾小球上皮，它们共同为选择性过滤分子提供了屏障。 
有兴趣适应该协议的研究人员应考虑以下因素：首先，根据所用干细胞系的固有生长特性，可?...

器官芯片设备是动态3D体外模型，采用分子和机械刺激以及血管化来形成组织 - 组织界面，模拟特定器官的结构和功能。先前建立的旨在概括肾脏肾小球（肾小球芯片）的器官芯片装置由动物细胞系1或异质来源的人类原代和永生化细胞系2，3组成。遗传异质细胞来源的使用呈现出差异，显着限制了对患者特异性反应和遗传学或疾病机制的研究4，5。应对这一挑战取决于来自具有保留分子和遗传谱的特定个体的同基因细胞系的可用性，以便为体外模型工程提供更准确的微环境2，3，6。由于人类iPS细胞培养的进步，现在可以很容易地产生人类来源的同基因细胞系。由于人iPS细胞通常是非侵入性来源的，可以无限期地自我更新，并分化成几乎任何细胞类型，因此它们可以作为建立体外模型（例如肾小球芯片7，8）的有吸引力的细胞来源。肾小球滤过屏障是血液滤过的主要部位。血液首先通过血管内皮，肾小球基底膜过滤，最后通过称为足细胞的特殊上皮。过滤屏障的所有三个组成部分都有助于分子的选择性过滤。这里介绍的是一种建立器官芯片装置的方案，该装置与来自单个人iPS细胞源的血管内皮和肾小球上皮连接。虽然该协议对于设计同基因和血管化芯片以概括肾小球滤过屏障特别有用，但它也为开发其他类型的个性化器官芯片和多器官平台（如同基因“身体芯片”系统）提供了蓝图。
本文描述的方案始于将人iPS细胞分化为两个独立的谱系 - 外中胚层和中胚层细胞，随后分别分化为血管内皮和肾小球上皮。为了产生侧中胚层细胞，将人iPS细胞接种在基底膜基质1包被板上，并在补充有Wnt激活剂CHIR 99021和有效的中胚层诱导剂骨形态发生4（BMP4）的N2B27培养基中培养3天（无培养基交换）。所得的外侧中胚层细胞先前通过短吻（T），混合配对样同源盒（MIXL）和子宫真皮素（EOMES）的表达来表征9。随后，将侧中胚层细胞在补充有VEGF165和佛司可林的培养基中培养4天，以诱导基于VE-钙粘蛋白和/或PECAM-1表达使用磁激活细胞分选（MACS）分选的血管内皮细胞。通过将所得血管内皮细胞（viEC）培养在基底膜基质3包被烧瓶上进行扩增，直到准备在微流体装置中接种。
为了产生中胚层细胞，将人iPS细胞接种在基底膜基质2包被板上，并在含有Activin A和CHIR99021的培养基中培养2天。所得中胚层细胞的特征在于HAND1，鹅肌和短肠（T）的表达，如前所述2，10，11。为了诱导中间中胚层（IM）细胞分化，将中胚层细胞在补充有BMP-7和CHIR99021的培养基中培养14天。由此产生的IM细胞表达Wilm肿瘤1（WT1），配对盒基因2（PAX2）和奇数跳跃相关蛋白1（OSR-1）2，10，11。
设计了一种基于双通道聚二甲基硅氧烷（PDMS）的微流控芯片，以概括 体外肾小球滤过屏障的结构。尿道为 1，000 μm x 1，000 μm（宽 x 高），毛细血管通道尺寸为 1，000 μm x 200 μm（宽 x 高）。流体通道两侧的空心室促进了循环拉伸和松弛循环。将细胞接种到分离尿路和毛细血管通道的柔性PDMS膜（50μm厚）上。该膜配备有六角形孔（直径7μm，相距40μm），以帮助促进细胞间信号传导（图1A）2，12。在IM诱导完成前两天，微流控芯片涂覆基底膜基质2。在IM诱导完成前1天，使用内皮维持培养基将viEC接种到微流控芯片的毛细血管通道中，并将芯片倒置以使细胞粘附在ECM涂层的PDMS膜的基底侧。在IM诱导完成的当天，使用补充有BMP7，Activin A，CHIR99021，VEGF165和 所有反式 维甲酸的培养基将细胞接种到微流控芯片的尿通道中，以诱导芯片内的足细胞分化。第二天，将足细胞诱导培养基和内皮维持培养基填充培养基，并将0.4 Hz和流体流量（60 μL / h）下的10%机械应变施加到芯片上。
使用足细胞诱导培养基（在尿通道中）和内皮维持培养基（在血管通道中）将细胞化微流控芯片再培养5天。将所得肾小球芯片在足细胞和内皮细胞的维持培养基中再培养长达7天。分化的足细胞正表达谱系特异性蛋白，包括鬼臼素和肾单位13，14，而viECs正表达谱系鉴定蛋白PECAM-1和VE-钙粘蛋白，所有这些都是维持肾小球滤过屏障完整性的重要分子15，16.足细胞和viEC都被发现分泌最丰富的肾小球基底膜蛋白，胶原蛋白IV，这对组织的成熟和功能也很重要。
发现肾小球芯片中的过滤屏障的三组分系统 - 内皮，基底膜和上皮 - 选择性过滤分子并对化疗，肾毒性药物治疗作出反应。药物治疗结果表明，肾小球芯片可用于肾毒性研究和疾病建模。该协议为从同基因iPS细胞衍生物工程功能性微流控肾小球芯片提供了一般指南。研究人员可以根据需要进行工程芯片的下游分析。有关使用肾小球芯片模拟药物性肾小球损伤的更多信息，请参阅以前的出版物2，12。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies</td>
			<td>Thermo/Life Technologies</td>
			<td>A32744; A32754; A-11076; A32790; A21203; A11015</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Thermo/Life Technologies</td>
			<td>14-9871-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>Progen</td>
			<td>GP-N2</td>
			<td></td>
		</tr>
		<tr>
			<td>PECAM-1</td>
			<td>R&#38;D Systems</td>
			<td>AF806</td>
			<td></td>
		</tr>
		<tr>
			<td>Podocin</td>
			<td>Abcam</td>
			<td>ab50339</td>
			<td></td>
		</tr>
		<tr>
			<td>VE-Cadherin</td>
			<td>Santa Cruz</td>
			<td>sc-9989</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrices</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Fibronectin, Human</td>
			<td>Corning</td>
			<td>356008</td>
			<td>Basement membrane (3)</td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>Iwai North America</td>
			<td>N8922012</td>
			<td>Basement membrane matrix (2)</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Basement membrane matrix (1); may show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11 (Duke University clone #11) iPS cell line was generated at the Duke iPSC Core Facility and provided to us by the Bursac Lab at Duke University. The line has been tested and found to be free of mycoplasma (last test in November 2021) and karyotype abnormalities (July 2019)</td>
		</tr>
		<tr>
			<td>Culture medium growth factors and media supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo/Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from Bovine serum, Texas Red conjugate</td>
			<td>Thermo/Life Technologies</td>
			<td>A23017</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>Stem Cell Technologies</td>
			<td>72262</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>Thermo/Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement (50x) without Vitamin A</td>
			<td>Thermo/Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete medium kit with CultureBoost-R</td>
			<td>Cell Systems</td>
			<td>4Z0-500-R</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo/Life Technologies</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>Thermo/Life Technologies</td>
			<td>10565042</td>
			<td>DMEM/F12 with glutamine</td>
		</tr>
		<tr>
			<td>Forskolin (Adenylyl cyclase activator)</td>
			<td>Abcam</td>
			<td>ab120058</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo/Life Technologies</td>
			<td>35050061</td>
			<td>glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>Thermo/Life Technologies</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin solution</td>
			<td>Stem Cell Technologies</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Activin A</td>
			<td>Thermo/Life Technologies</td>
			<td>PHC9544</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP4</td>
			<td>Preprotech</td>
			<td>120-05ET</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>Thermo/Life Technologies</td>
			<td>PHC9544</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>Thermo/Life Technologies</td>
			<td>PHC9394</td>
			<td></td>
		</tr>
		<tr>
			<td>Inulin-FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F3272</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>Stem Cell Technologies</td>
			<td>05850</td>
			<td>Human iPS cell culture media (CCM). Add 5x supplement according to the manufacturer. Human iPS CCM can be stored for up to 6 months at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Thermo/Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Thermo/Life Technologies</td>
			<td>21103049</td>
			<td>Lateral mesoderm basal media</td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)</td>
			<td>Thermo/Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin, liquid (100x)</td>
			<td>Thermo/Life Technologies</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor (Y27632)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro-34 SFM</td>
			<td>Thermo/Life Technologies</td>
			<td>10639011</td>
			<td>Endothelial cell culture medium (CCM). Add supplement according to manufacturer. Endothelial CCM can be stored for up to two weeks at 4 &#176;C or -20 &#176;C for up to 6 months.</td>
		</tr>
		<tr>
			<td>TGF-Beta inhibitor (SB431542)</td>
			<td>Stem Cell Technologies</td>
			<td>72234</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymes and other reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Thermo/Life Technologies</td>
			<td>A1110501</td>
			<td>Cell detachment buffer</td>
		</tr>
		<tr>
			<td>Dimethyl Suloxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>VWR</td>
			<td>97065-058</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Thermo/Life Technologies</td>
			<td>28906</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile distilled water</td>
			<td>Thermo/Life Technologies</td>
			<td>15230162</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>97062-208</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 0.05%</td>
			<td>Thermo/Life Technologies</td>
			<td>25300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>(Orb) Hub module</td>
			<td>Emulate</td>
			<td>ORB-HM1</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm x 15 mm round petri dish</td>
			<td>Fisherbrand</td>
			<td>FB087579B</td>
			<td></td>
		</tr>
		<tr>
			<td>120 x 120 mm square cell culture dish</td>
			<td>VWR</td>
			<td>688161</td>
			<td></td>
		</tr>
		<tr>
			<td>Accuri C6</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>Corning</td>
			<td>29442-462</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating Unit</td>
			<td>SP Bel-Art</td>
			<td>F19917-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B99516</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>Thermo/Life Technologies</td>
			<td>AMF7000</td>
			<td>Fluorescent microscope to take images of fixed and stained cells.</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>100503-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>Thermo/Life Technologies</td>
			<td>51030403</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipeed with AxioCam 503 camera</td>
			<td>Carl Zeiss Micrscopy</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>VWR</td>
			<td>40101-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>VWR</td>
			<td>21905-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Leoca SP8 Upright Confocal Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Media reservoir (POD Portable Module)</td>
			<td>Emulate</td>
			<td>POD-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td>VWR</td>
			<td>12620-926</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ-chip</td>
			<td>Emulate</td>
			<td>S-1 Chip</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ-chip holder</td>
			<td>Emulate</td>
			<td>AK-CCR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>Denville Scientific</td>
			<td>P1096-FR</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>Denville Scientific</td>
			<td>P1125</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>Denville Scientific</td>
			<td>P1121</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>Denville Scientific</td>
			<td>P1122</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>Denville Scientific</td>
			<td>P1122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Asher</td>
			<td>Quorum tech</td>
			<td>K1050X RF</td>
			<td>This Plasma Etcher/Asher/Cleaner was used as a part of Duke University&#39;s Shared Materials Instrumentation Facility (SMiF).</td>
		</tr>
		<tr>
			<td>Round bottom polystyrene test tube with cell strainer snap cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, indivdually wrapped</td>
			<td>Corning</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, indivdually wrapped</td>
			<td>Corning</td>
			<td>356525</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, indivdually wrapped</td>
			<td>Corning</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#181;m, PES</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge tubes</td>
			<td>Thomas Scientific</td>
			<td>1138W14</td>
			<td></td>
		</tr>
		<tr>
			<td>T75cm2 cell culture flask with vent cap</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture-treated 12 well plates</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture-treated 6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum modulator and perstaltic pump (Zoe Culture Module)</td>
			<td>Emulate</td>
			<td>ZOE-CM1</td>
			<td>Organ Chip Bioreactor</td>
		</tr>
		<tr>
			<td>VE-Cadherin CD144 anti-human antibody - APC conjugated</td>
			<td>Miltenyi Biotec</td>
			<td>130-126-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD144 MicroBeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-857</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 MicroBead Kit, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-935</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
	</tbody>
</table>

isogenic kidney glomerulus chip engineered from human induced pluripotent stem cells

慢性肾病（CKD）影响了15%的美国成年人口，但由于缺乏能够准确预测人类生物反应和肾毒性的功能模型，靶向治疗的建立受到限制。肾脏精准医学的进步可以帮助克服这些限制。然而，先前建立的人肾小球体 外 模型 - 血液过滤的主要部位和许多疾病和药物毒性的关键靶标 - 通常采用功能特征有限和不匹配的遗传背景的异质细胞群。这些特征极大地限制了它们在患者特异性疾病建模和治疗发现中的应用。 
本文提出了一种方案，该方案整合了来自单个患者的人诱导多能干（iPS）细胞来源的肾小球上皮（足细胞）和血管内皮，以设计同基因和血管化的微流体肾小球芯片。由此产生的肾小球芯片由干细胞衍生的内皮和上皮细胞层组成，它们表达谱系特异性标志物，产生基底膜蛋白，并形成类似于肾脏肾小球滤过屏障的组织 - 组织界面。工程化的肾小球芯片选择性地过滤分子并概括药物引起的肾损伤。使用同基因细胞类型重建肾小球结构和功能的能力为具有患者特异性的肾脏疾病建模创造了机会，并推进了器官芯片在肾脏精准医学和相关应用中的实用性。

在这里，我们表明肾小球的功能3D体外模型可以从人iPS细胞的等基因来源血管化和上皮化。具体来说，该协议提供了有关如何应用人类iPS细胞技术的说明，特别是它们分化成特殊细胞类型的能力，以产生肾小球上皮（足细胞）和血管内皮（viEC），可以与微流体装置集成以模拟患者特定水平的人肾的结构和功能。该协议和时间表的示意图（图1A）描述了如何培养有丝分裂活性的人iPS细胞（图1B...

Watch this Scientific Journal Video about Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells at JoVE.com

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

这里介绍的是一种设计个性化器官芯片系统的协议，该系统通过整合与人类诱导的多能干细胞分化的遗传匹配的上皮和血管内皮细胞来概括肾小球过滤屏障的结构和功能。这种生物工程系统可以推进肾脏精准医疗和相关应用。

由人诱导多能干细胞改造的同基因肾小球芯片

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

同基因肾小球芯片将使患者特定的疾病建模和肾毒性测试以及先进的精准医学应用成为可能。肾小球上皮和内皮来源于单个人诱导的多能干细胞或IPSC。IPSC具有无限的自我更新，几乎可以分化成任何细胞类型。该技术可用于评估病理细胞表型和疾病。患者特异性器官芯片也可以作为药物筛选和机制研究的平台。该模型可应用于针对特定患者个性化芯片系统的开发。该系统有可能在临床试验之前预测患者特定的反应。首先将25微升基底膜基质2溶液轻轻加入尿通道，20微升加入芯片的毛细血管通道。取两个无菌的15毫升锥形管盖，并用500微升无菌蒸馏水填充它们。将盖子放在培养皿中以防止干燥，并将盖子放在培养皿上。在 37 摄氏度下孵育过夜。继续，从含有15天血管内皮细胞培养物的T75烧瓶中吸出培养基，浓度为90%。加入五毫升细胞分离缓冲液，并在 37 摄氏度下孵育 5 至 7 分钟。然后将细胞转移到15毫升锥形管中，并加入5毫升DMEM / F-12。以200倍G离心五分钟。除去上清液并将细胞重悬于300微升内皮维持培养基中。用血细胞计数器计数细胞以获得大约200万个细胞。使用 P200 屏障尖端，用 200 微升 DMEM/F-12 冲洗微流控芯片的顶部和底部通道，同时从出口外围吸出。将连接到吸气器的 P200 屏障尖端牢固地握住芯片，远离底部通道的出口。将20微升血管内皮细胞悬液牢固地注入芯片的毛细血管通道中，并小心地从出口外围吸出培养基。在显微镜下检查是否有气泡或不均匀的播种密度。轻轻倒置芯片，使细胞可以粘附在柔性PDMS膜的基底侧。将芯片放入支架盒中，并在盒中加入三毫升PBS，以防止膜干燥。将芯片在 37 摄氏度下孵育三个小时。检查显微镜下的底部通道，以确定附着在柔性PDMS膜上的细胞汇合层。通过在底部通道的入口处添加200微升内皮维持培养基来洗涤未连接的细胞，同时从毛细血管通道出口的外围吸出。将芯片放回支架盒中，并在 37 摄氏度下孵育过夜。第二天，用200微升内皮维持培养基轻轻冲洗毛细血管通道。用 200 微升 DMEM/F-12 冲洗尿道，并在入口和出口处滴下 50 微升 DMEM/F-12。进行中间中胚层细胞，用一毫升胰蛋白酶EDTA替换12孔板每个孔中的中间中胚层诱导培养基，并在37摄氏度下孵育五分钟。使用细胞提升器轻轻刮取细胞，并使用移液解离细胞。向每个孔中加入两毫升胰蛋白酶中和溶液，并将细胞转移到50毫升锥形管中。用DMEM / F-12将细胞悬液的体积补足至50毫升，并以200倍G离心五分钟。吸出上清液并将细胞重悬于500微升中间胚层诱导培养基中，以获得约300万个细胞。用血细胞计数器计数细胞。握住芯片并将25微升细胞悬液牢固地注入芯片的尿道中，同时小心地从出口外围吸出培养基。检查气泡或不均匀的细胞接种密度。将三毫升PBS加入芯片支架盒中，并在37摄氏度下孵育三小时。孵育后，用200微升各自的细胞培养基冲洗两个通道。将空的 P200 屏障尖端连接到尿道和毛细血管通道的两个出口。移取200微升内皮维持培养基，并将其一半注入毛细血管通道入口。释放入口内的移液器吸头，使通道的入口和出口都连接到充满介质的移液器吸头上。用200微升中间中胚层维持培养基对尿道入口重复此过程。将芯片与嵌入在 37 摄氏度下的尖端孵育过夜。要将芯片连接到器官芯片生物反应器，首先，从通道中取出P200尖端。将相应介质的液滴添加到尿道和毛细血管通道的入口和出口处，以防止干燥。向尿入口储液器中加入 3 毫升温热的足细胞诱导培养基，向毛细血管入口储液器中加入 3 毫升温热的内皮维持培养基。将 300 微升相应培养基直接添加到出口端口上方的出口储液罐中。将豆荚滑到托盘上并进入器官芯片生物反应器。使用旋转拨盘选择并开始黄金周期两分钟。目视检查吊舱底部是否有所有四个流体端口上的小液滴。要实现吊舱底部和微流控芯片端口之间的流体间接触，请将芯片载体轻轻滑入吊舱。轻轻向上按切屑托架卡舌。从芯片表面吸出多余的介质。将器官芯片生物反应器的流速设置为每小时 60 微升。将循环应变设置为 10%，频率为 0.4 赫兹。使用器官芯片生物反应器上的旋转拨盘选择调节循环并运行两个小时。目视检查出口储液罐是否增加介质液位。使用器官芯片生物反应器上的旋转拨盘选择调节循环。培养第 17 天后，每天从远离端口斜对角线的尿道出口储液器吸出培养基，但在储液器中保留一些培养基。每两天用最多三毫升足细胞诱导培养基补充尿道入口储液器，持续五天。五天后，从尿道吸出培养基，但在储液器中保留一些培养基。每天用三毫升足细胞维持培养基补充尿道入口储液器。同样，从毛细血管通道出口吸出培养基，并每天用内皮维持培养基补充入口储液器。使用该协议，人诱导的多能干细胞用于通过繁殖血管内皮细胞和足细胞来开发肾小球的功能体外模型。在足细胞诱导和血管内皮增殖后第21天，芯片内的细胞表达谱系识别标志物，例如足细胞的Podocin和Nephine，血管内皮细胞的VE Cadherin和PECAM-1。足细胞和血管内皮细胞层均表达胶原蛋白IV，这是肾小球中最丰富的GBM蛋白。肾小球芯片选择性地过滤来自毛细血管的小分子，如菊粉进入尿通道，同时阻止白蛋白等大蛋白离开毛细血管通道，显示出选择性分子过滤功能。在第 4 至 7 天的内皮诱导期间，细胞数量的增加可能导致细胞的第二层，但过度接种可能会导致聚集，从而阻碍分化。在PDMS芯片组件粘合不充分、流体流动路径堵塞或过滤屏障受损的情况下，可能会观察到尿道和毛细血管通道之间的意外流体交叉流动。从第 15 天开始播种内皮和上皮细胞并维护芯片时，重要的是在每一步目视检查通道中的气泡。在等基因肾小球芯片中，可以施用临床相关的候选药物来测试其治疗潜力或毒性水平。这项技术现在为了解人类肾脏疾病的遗传基础和开发个性化疗法提供了机会。

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

从人体 iPS 细胞中提取的网状工程心脏组织用于活体心肌修复

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

从多能干细胞生成人体血管类器官

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...