Name: creation of a knee joint-on-a-chip for modeling joint diseases and testing drugs
Uploaded: 2023-01-27T12:30:00
Description: Watch this Scientific Journal Video about Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs at JoVE.com

这项研究主要由美国国立卫生研究院（UG3 / UH3TR002136，UG3 / UH3TR003090）资助。此外，我们感谢Paul Manner博士（华盛顿大学）提供人体组织样本，感谢Jian Tan博士帮助分离MSCs和创建细胞池。

以下协议遵循匹兹堡大学和匹兹堡大学人类研究伦理委员会的伦理准则。有关本研究所用材料的信息列在 材料表中。
1. 制造3D打印生物反应器
<ol><li>使用计算机软件设计骨软骨（图2A）和微型关节生物反应器（图2B），包括腔室，插入物和盖子。每个零件的尺寸信息如图 S1所示。</li>
	<li>将设...

<ol>
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Part C, Methods. 27 (2), 124-138 (2021).</li><li>He, 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=Pathogenesis+of+osteoarthritis:+risk+factors,+regulatory+pathways+in+chondrocytes,+and+experimental+models.">Pathogenesis of osteoarthritis: risk factors, regulatory pathways in chondrocytes, and experimental models.</a> Biology. 9 (8), 194 (2020).</li><li>Ronaldson-Bouchard, K., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-a-chip:+A+fast+track+for+engineered+human+tissues+in+drug+development.">Organs-on-a-chip: A fast track for engineered human tissues in drug development.</a> Cell Stem Cell. 22 (3), 310-324 (2018).</li><li>Lin, H., Sohn, J., Shen, H., Langhans, M. T., Tuan, R. 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在本文中，我们提出了一种用于创建膝关节芯片系统的协议，其中骨骼，软骨，脂肪组织和滑膜样组织由MSC形成并在定制的生物反应器中共同培养。这种具有即插即用功能的多组分人类细胞衍生系统是研究关节疾病发病机制和开发药物的新工具。 
鉴于不同的组织有利于特定的培养基，因此为每个组织提供相应的培养基并防止流动之间的自由培养基交换至关重要。特别是，在?...

骨关节炎 （OA） 等关节疾病非常普遍且使人衰弱，是全球残疾的主要原因1。据估计，仅在美国，OA就影响了2700万患者，12.1%的60岁及以上成年人2。不幸的是，目前用于治疗关节疾病的大多数药物都是姑息性的，并且没有有效的疾病改善OA药物（DMOADs）可用3。这种未满足的医疗需求主要源于缺乏研究疾病机制和开发潜在DMOAD的有效模型。传统的二维（2D）细胞培养不能反映关节组织的3D性质，并且组织外植体的培养经常受到显着细胞死亡的阻碍，并且通常无法复制动态组织互连4。此外，遗传和解剖学差异显着降低了动物模型的生理相关性4。
器官芯片（OoCs）或微生理系统是工程，生物学和医学界面的一个有前途的研究领域。这些 体外 平台是最小的功能单位，可复制其 体内 对应物的定义健康或病理特征5。此外，这些小型化系统可以容纳不同的细胞和基质，并模拟不同组织之间的生物物理和生化相互作用。因此，能够忠实地概括天然滑膜关节的微生理系统有望为关节疾病建模和开发潜在的DMOAD提供有效的平台。
人间充质干细胞（MSCs）可以从全身的许多组织中分离出来，并分化为成骨、软骨和成脂谱系6。间充质干细胞已成功用于设计各种组织，包括骨骼，软骨和脂肪组织6，这意味着它们代表了设计膝关节组织成分的有前途的细胞来源。我们最近开发了一种微型关节模拟微生理系统，名为miniJoint，包括MSC衍生的骨骼，软骨，纤维和脂肪组织7。特别是，新颖的设计可以通过微流体流动或渗透实现组织串扰（图1）。在这里，我们提出了芯片组件的制造，组织组件的工程，芯片中工程组织的培养以及用于下游分析的组织收集的协议。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64186/64186fig01.jpg"> 图 1：miniJoint 芯片示意图，显示了不同组织组件和介质流的排列。 OC = 骨软骨组织。 <a href="https://www.jove.com/files/ftp_upload/64186/64186fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma -Aldrich</td>
			<td>I17018-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well non-tissue culture plate</td>
			<td>Corning Falcon&#174; Plates</td>
			<td>351146</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well non-tissue culture plate</td>
			<td>Corning Falcon&#174; Plates</td>
			<td>351147</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL syringes</td>
			<td>BD Syringe Luer Lock Cascade Health</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>EK Industries</td>
			<td>1198</td>
			<td>1% w/v, pH 1.0</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491-015</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM</td>
			<td>Gibco</td>
			<td>12571-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Integra Miltex</td>
			<td>12-460-407</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY&#174; fluorophore</td>
			<td>Molecular Probes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone morphogenic protein 7 (BMP7)</td>
			<td>Peprotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fisher Brand</td>
			<td>16100110</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
		</tr>
		<tr>
			<td>Dexmethasome</td>
			<td>Sigma -Aldrich</td>
			<td>02-05-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Shell 450 photopolymer in</td>
			<td>EnvisionTec</td>
			<td>RES-01-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini-Bio Products</td>
			<td>900-208</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>3505-061</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin from bovine skin</td>
			<td>Hyclone</td>
			<td>1003372809</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Sigma -Aldrich</td>
			<td>SH30588.02</td>
			<td></td>
		</tr>
		<tr>
			<td>indomethacin</td>
			<td>Sigma -Aldrich</td>
			<td>I7378-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS)</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>interleukin 1&#946;</td>
			<td>Peprotech</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Leur-loc connectors</td>
			<td>Cole-Parmer Instruments</td>
			<td>45508-50</td>
			<td></td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma -Aldrich</td>
			<td>115388-93-7</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma -Aldrich</td>
			<td>1003129352</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium bags</td>
			<td>KiYATEC</td>
			<td>FC045</td>
			<td></td>
		</tr>
		<tr>
			<td>Methacrylic Anhydride</td>
			<td>Sigma -Aldrich</td>
			<td>102378580</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered Saline</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pointed forceps</td>
			<td>Fisher Brand</td>
			<td>12000122</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon mold</td>
			<td>McMaster-Carr</td>
			<td>RC00114P</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon o-rings</td>
			<td>McMaster-Carr</td>
			<td>ZMCCs1X5</td>
			<td>1mm x 5mm</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault Syst&#232;mes SE, V&#233;lizy-Villacoublay, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blades</td>
			<td>Integra Miltex</td>
			<td>4-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Lagato210P, KD Scientific</td>
			<td>Z569631</td>
			<td>10 syringe racks</td>
		</tr>
		<tr>
			<td>T-182 tissue culture flasks</td>
			<td>Fisher Brand</td>
			<td>FB012939</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 150 mm</td>
			<td>Fisher Brand</td>
			<td>FB012925</td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor Beta (TGF-&#946;3)</td>
			<td>Peprotech</td>
			<td>100-36E</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Flashlight</td>
			<td>KBS</td>
			<td>KB70109</td>
			<td>395 nm</td>
		</tr>
		<tr>
			<td>Vida Desktop 3D Printer</td>
			<td>EnvisionTec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin D3</td>
			<td>Sigma -Aldrich</td>
			<td>32222-06-3</td>
			<td>1,25-dihydroxyvitamin D3</td>
		</tr>
	</tbody>
</table>

creation of a knee joint-on-a-chip for modeling joint diseases and testing drugs

骨关节炎（OA）等使人衰弱的关节疾病的高患病率构成了很高的社会经济负担。目前，针对关节疾病的可用药物大多是姑息性的。对有效的疾病修饰OA药物（DMOADs）的需求未得到满足，主要是由于缺乏适当的模型来研究疾病机制和测试潜在的DMOADs。在这里，我们描述了一个微型滑膜关节模拟微生理系统（miniJoint）的建立，该系统包括来自人间充质干细胞（MSCs）的脂肪，纤维和骨软骨组织成分。为了获得三维（3D）微组织，在分化之前或之后将MSCs包封在可光交联的甲基丙烯酸化明胶中。然后将充满细胞的组织结构整合到3D打印的生物反应器中，形成miniJoint。引入单独的成骨、纤维化和成脂培养基流以维持各自的组织表型。通过软骨、滑膜和脂肪组织灌注常见的共享流，以实现组织串扰。这种流动模式允许在一个或多个组织成分中诱导扰动以进行机理研究。此外，可以通过所有培养基流的“全身给药”或通过仅将药物添加到共享的“滑液”模拟流中的“关节内给药” 来 测试潜在的DMOAD。因此，miniJoint可以作为一个多功能 的体外 平台，用于有效研究疾病机制和测试个性化医疗中的药物。

收集mini关节的所有组织以分析其在mini关节中培养28天后的表型（图4A）。这在我们之前的出版物7中已经详细介绍了。
通过使用RT-qPCR、免疫染色和组织学染色，证实单个微组织的组织特异性表型得到了很好的维持（图4）。例如，OC微组织的骨成分（OC-O）而不是其他组织成分表达高水平的骨钙素（OCN）。相?...

Watch this Scientific Journal Video about Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs at JoVE.com

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

我们提供了从人间充质干细胞生成四种类型的组织的详细方法，这些组织用于概括人膝关节中的软骨、骨骼、脂肪垫和滑膜。这四个组织被集成到一个定制的生物反应器中，并通过微流体连接，从而产生一个膝关节芯片。

创建膝关节芯片，用于关节疾病建模和药物测试

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

由于缺乏生理相关的临床前模型，我们在临床试验期间准确预测人类对药物反应的能力受到限制。这种微生理系统可以解决这种差异。这种人类细胞衍生系统准确地模拟了人类膝关节骨关节炎的整个关节疾病性质，允许在临床试验之前测试疾病修饰骨关节炎药物，可能节省数百万美元。开始制备甲基丙烯酸化明胶或GelMA，将17克B型明胶添加到500毫升蒸馏水中。在37摄氏度的摇床上混合30分钟，加入13毫升甲基丙烯酸酐，然后在摇床上混合过夜。第二天，将大约 60 毫升的 GelMA 等分试样放入透析袋中。将所有透析袋放入带搅拌棒的蒸馏水中七天。每天多次换水，并将袋子放在四摄氏度下过夜。在第 7 天，将 GelMA 倒入培养皿中，并在冻干前将其冷冻在零下 80 摄氏度。然后，通过将GelMA置于冻干机的真空室中来冻干。在从冻干机中取出之前，请确保完全干燥。接下来，将冻干的GelMA溶解在15%浓度的Hanks平衡盐溶液中，并使用少量氢氧化钠将pH调节至7.4。根据获得的体积，用抗生素抗真菌剂和0.15%苯基次膦酸锂或LAP补充溶液。保护GelMA溶液免受光照，并将其储存在零下20摄氏度，直到进一步使用。高压灭菌器3D打印的双流生物反应器室，盖子和插入物在121摄氏度的高压灭菌袋中，用蒸汽加热20分钟。然后，用干热20分钟。灭菌后，将生物反应器室，盖子和插入物浸泡在生物安全柜内的15毫升无菌PBS中过夜，然后完全干燥。使用1000微升移液器，将每毫升细胞重悬20次10至第六个人骨髓来源的间充质干细胞或MSCs，在15%GelMA溶液中。然后，使用无菌手套将无菌干燥的硅胶模具压在培养皿上。用镊子将一个嵌件放入硅胶模具的每个孔中，将嵌件的孔侧朝下。完成后，使用200微升移液器在每个插入物中加入约50微升细胞悬液。通过将顶部暴露在 395 纳米波长的紫外线手电筒下 1.5 分钟，然后照亮另一侧 30 秒，将插入物内的凝胶交联。使用无菌镊子，立即将插入物转移到非组织培养六孔板中的8毫升生长培养基中，并使细胞在夜间恢复。为了开始脂肪组织的分化，将插入物转移到8毫升的成脂培养基中，并每天更换培养基28天。为了设计骨软骨单元，将插入物放入双流生物反应器室中，盖上孔盖，并以每分钟5微升的流速分别注入35毫升成骨培养基和35毫升成骨培养基。通过对相应的培养基类型进行每两周一次的注射器更换，将细胞维持分化28天。为了获得成纤维细胞，在含有20毫升纤维化培养基的T 150平方厘米组织细胞培养瓶中分化2D培养中的MSCs，持续21天。保持媒体的每周更换。21天后，使用四毫升胰蛋白酶分离细胞。将3D凝胶封装在插入物中。要获得滑膜样纤维组织或SFT，请遵循类似的方案。将内径为0.062英寸和外径为0.125英寸的硅胶管连接到一端的微型接头生物反应器杆和另一端的F 1/16诱饵锁连接器。然后，将3D微型接头生物反应器以及硅胶管和盖子高压灭菌。准备35毫升用于微型接头培养的培养基并将其装入培养基储液槽中。使用直镊子将骨软骨单元从双流生物反应器转移到微型关节生物反应器的右孔中。将脂肪组织插入物和纤维组织插入物分别转移到左孔和中间孔中，然后用灭菌盖覆盖所有孔。将微型接头夹入口连接到介质储液罐，将出口连接到注射器。然后，将注射器安装到注射泵上。将泵和薯片转移到培养箱中。将培养基储液槽放在培养箱外的冰上。以抽出模式操作泵，将介质从介质储液器吸入微型联合生物反应器室。继续这种微型关节培养过程28天。为了模拟关节炎症和软骨变性，以每毫升10纳克的速度将白细胞介素一个β添加到纤维化培养基菌株中，持续7天。确保在第三天更换注射器，然后再向纤维化培养基提供新鲜的白细胞介素一个β。对于药物测试，在白细胞介素一次β治疗三天后，在共享培养基中给药，模拟关节内给药，或在所有培养基类型中模拟全身给药。白介素一次β治疗七天后，收集单个组织进行分析。使用无菌镊子取出插入物后，将活检冲头推入插入物的中心以取出凝胶并将凝胶放入PBS中。接下来，在评估基因表达的同时将骨软骨凝胶切成两半。从每个培养基源收集约1.5毫升培养基，以14， 000 G离心10分钟。丢弃沉淀物后，将调节介质快速冷冻在液氮中。对于组织学染色和免疫染色，首先，将骨软骨和滑膜样纤维组织或SFT样品固定在10%缓冲的福尔马林中，并在升序浓度的乙醇中脱水。在二甲苯中清除样品，将它们嵌入石蜡中，最后将样品切成六微米厚的厚度。对于脂肪组织，用油红O溶液或BODIPY直接染色10%缓冲的正式和固定样品。单个微组织的组织特异性表型保持良好。在培养28天后收集微型关节的所有组织以分析其表型。骨软骨单位微组织的骨成分表达高水平的骨钙素。在骨软骨的软骨部分，组织表达明显高水平的二型胶原和聚集胶。代表性脂肪基因脂联素和瘦素在脂肪组织中的表达水平较高。钙矿物质、碱性磷酸酶和骨钙素蛋白的沉积主要见于骨软骨微组织的骨骼大小。骨软骨组织的软骨侧显示出良好的糖胺聚糖和二型胶原蛋白保留。培养28天后，软骨细胞肥大相关标志物10型胶原和印度刺猬的表达显着下调。在脂肪组织中发现丰富的脂滴沉积。免疫荧光染色显示，经过4周的培养，SFT对润滑素和钙粘蛋白11的表达非常稳定。在疾病模型中，IL one β处理导致SFT中的细胞凋亡和MMP-13水平升高。软骨降解用IL one β处理，提示骨软骨组织和SFT之间发生串扰。萘普生全身给药的治疗效果显示IL one β微型关节软骨降解减少。实时荧光定量PCR表明，四种潜在的疾病修饰骨关节炎药物部分逆转了软骨损失。该过程最重要的步骤包括插入物中凝胶的交联和插入物放入生物反应器中。该芯片允许调查骨关节炎中提供的其他风险因素的贡献，例如肥胖和机械损伤。它也可以用来研究其他关节疾病。

Research

JoVE Journal

Bioengineering

Plasma Lithography Surface Patterning for Creation of Cell Networks

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

病人的具体建模之心：的心室纤维方向估计

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

建立了外科手术引起的小鼠模型，以调查的progranulin在骨性关节炎的保护作用

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

对于膝下义肢的估算惯性振荡性质与反应板技术

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

创建患者专用硅胶心脏模型，应用于术前计划和实践培训

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...