在微加工微流体平台中生成简化的三维芯片皮肤模型
Summary
在这里，我们提出了一个协议，使用微加工微流体平台生成三维简化和无差别皮肤模型。平行流动方法允许真皮隔室 的原位 沉积，以便在顶部接种上皮细胞，所有这些都由注射器泵控制。
Abstract
这项工作提出了一种新的，具有成本效益的，可靠的微流体平台，具有产生复杂多层组织的潜力。作为概念证明，已经对包含真皮（基质）和表皮（上皮）隔室的简化和无差别的人体皮肤进行了建模。为了实现这一目标，已经开发了一种多功能且坚固耐用的乙烯基器件，分为两个腔室，克服了基于聚二甲基硅氧烷（PDMS）的微流体器件在生物医学应用中存在的一些缺点，例如使用昂贵和专用的设备或吸收小的疏水分子和蛋白质。此外，还开发了一种基于平行流动的新方法，使真皮和表皮隔室 的原位 沉积成为可能。皮肤结构由含有人原代成纤维细胞的纤维蛋白基质和播种在上面的永生化角质形成细胞的单层组成，随后在动态培养条件下维持。这种新的微流体平台开辟了模拟人类皮肤病并推断产生其他复杂组织的方法的可能性。
Introduction
最近，在开发和生产用于分析化妆品和药品毒性的体外人体皮肤模型方面取得了进展1。制药和护肤行业的研究人员一直在使用动物，小鼠是最常见的，来测试他们的产品2，3，4，5。然而，在动物身上测试产品并不总是预测人类的反应，这经常导致药物失败或对人类的不良反应，从而导致经济损失5，6。英国是1998年第一个禁止使用动物进行美容测试的国家。后来，在2013年，欧盟禁止在动物身上测试和批准化妆品（欧盟化妆品法规第1223/2009号）7。
其他国家也在考虑这一禁令，例如美国的”人道化妆品法案“8。除了道德问题之外，动物和人类皮肤之间的解剖学差异使得动物试验耗时，昂贵且通常无效。此外，到2025年，全球 体外 毒理学检测市场规模预计将达到269.8亿美元9。由于这些原因，需要为这些 体外 研究开发新的方法和替代方案，例如生物工程人体皮肤模型，以便在不使用动物的情况下测试化妆品和药物的安全性和毒性作用。
市售有两种不同的体外，人体皮肤模型。第一种类型由分层的表皮当量组成，这些表皮当量含有多层分化的角质形成细胞，这些细胞接种在不同的材料上。其中一些已获得经济合作与发展组织（OECD）的批准，并得到（欧洲替代方法验证中心（ECVAM））的验证，用于皮肤腐蚀和刺激测试，例如EpiDerm或SkinEthic10，11，12。第二种类型是全皮肤等效物，具有一层分化的人类角质形成细胞，这些角质形成细胞接种在含有成纤维细胞的三维（3D）支架上，例如T-Skin和EpiDerm-FT。然而，这些模型是在静态条件下培养的，这使得它们无法准确表示人体生理条件。
最近的兴趣集中在以动态灌注13，14，15，16，17，18，19的细胞插入（CCI）格式生成体外3D皮肤模型。然而，根据它们在该领域的经典定义，这些系统不能严格意义上被视为微流体的芯片皮肤。Ingber对芯片上器官的定义指出，器官必须放置在微流体通道内，这是只有少数设备满足20，21的条件。到目前为止，芯片上的皮肤已经将大部分简单的上皮模拟为由多孔膜隔开的单细胞层和/或真皮细胞层22，23。虽然在微流体系统中对皮肤建模有一些进展16，24，但目前没有文献显示一个符合Ingber定义的芯片器官系统，能够在原位产生多层皮肤，包括上皮和基质成分。
在这项工作中，提出了一种新的，经济高效，强大的，基于乙烯基的微流体平台，用于芯片皮肤应用。该平台通过微加工生产，这为制造过程提供了更多的简单性，并在设备布局中增加了灵活性和多功能性，克服了PDMS25的一些局限性 。还设计了一种通过注射泵控制的平行流量引入简化的皮肤结构的方法。平行流动允许两种粘度非常不同的流体（在这种情况下是缓冲液和纤维蛋白预凝胶）通过通道灌注而不相互混合。作为概念证明，在装置中引入了含有嵌入在模仿真皮的纤维蛋白基质中的成纤维细胞的真皮表皮构建体，在其上加载单层角质形成细胞以模拟未分化的表皮。真皮隔室高度可以通过改变流速来调节。与之前描述的模型22，26，27，28，29相比，这项工作的主要新颖之处在于通过微流体在微室内开发3D结构。虽然本文提出了一种简化的未分化皮肤，但长期目标是生成和表征完全分化的皮肤结构，以证明其在药物和化妆品测试目的中的可行性和功能。
Protocol
Representative Results
Discussion
开发这种方法的动机是希望在高通量平台中模拟皮肤病并研究新的创新疗法的效果。迄今为止，该实验室通过手动或借助3D生物打印技术将纤维蛋白凝胶与成纤维细胞一起投射到细胞培养插入板中并在其上接种角质形成细胞来生产这些真皮表皮等效物。一旦角质形成细胞达到汇合，3D培养物暴露于气液界面，其诱导角质形成细胞分化，产生分层的表皮，从而完全发育出圆周间人皮肤32，33，34。<sup c…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们衷心感谢哈维尔·罗德里格斯博士、玛丽亚·路易莎·洛佩斯博士、卡洛斯·马特兰和胡安·弗朗西斯科·罗德里格斯提供的非常有用的建议、讨论和/或初步数据。我们也衷心感谢Sergio Férnandez，Pedro Herreros和Lara Stolzenburg对这个项目的贡献。特别感谢Marta García博士获得GFP标签的hFBs和hKCS。最后，我们承认吉列尔莫·比斯凯诺和安吉利卡·科拉尔的出色技术援助。这项工作得到了”马德里社区投资集团活动计划”，项目S2018/BAA-4480，Biopieltec-CM的支持。这项工作也得到了”Excela de excelencia”项目EPUC3M03，CAM的支持。CONSEJERÍA DE EDUCACIÓN E INVESTIGACIÓN.
Materials
| Amchafibrin | Rottafarm | Tranexamic acid | |
| Antibiotic/antimycotic | Thermo Scientific HyClone | ||
| Calcium chloride | Sigma Aldrich | ||
| Culture plates | Fisher | ||
| DMEM | Invitrogen Life Technologies | ||
| Double-sided tape vynil | ATP Adhesive Systems | GM 107CC, 12 µm thick | |
| Edge plotter | Brother | Scanncut CM900 | |
| FBS | Thermo Scientific HyClone | ||
| Fibrinogen | Sigma Aldrich | Extracted from human plasma | |
| Glass slide | Thermo Scientific | ||
| GFP-Human dermal fibroblasts | – | Primary. Gift from Dr. Marta García | |
| H2B-GFP-HaCaT cell line | ATCC | Immortalized keratinocytes. Gift from Dr. Marta García | |
| Live/dead kit | Invitrogen | ||
| PBS | Sigma Aldrich | ||
| Polycarbonate membrane | Merk TM | 5 µm pore size | |
| Polydimethylsiloxane | Dow Corning | Sylgard 184 | |
| Sodium chloride | Sigma Aldrich | ||
| Syringes | Terumo | 5 mL | |
| Thrombin | Sigma Aldrich | 10 NIH/vial | |
| Transparent adhesive vinyl | Mactac | JT 8500 CG-RT, 95 µm thick | |
| Trypsin/EDTA | Sigma Aldrich | ||
| Tubing | IDEX | Teflon, 1/16” OD, 0.020” ID |
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