Name: generation of a simplified three-dimensional skin-on-a-chip model in a micromachined microfluidic platform
Uploaded: 2021-05-17T14:30:00
Description: Watch this Scientific Journal Video about Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform at JoVE.com

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.

1. 芯片设计及微加工参数
<ol><li>使用FreeCAD开源设计软件设计微流控芯片层;有关通道的尺寸，请参阅 表1。 在设计中包括四个直径为 2.54 mm 的孔，以使用定制的对准器实现正确的层叠加。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>长度（微米）</td>
			<td>宽度（微米）</td>
		</tr>
<tr>
<td>下腔室</td>
			<td>28,400</td>
			<td>800</td>
		</tr>
<...

<ol>
	<li>McNamee, 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=A+tiered+approach+to+the+use+of+alternatives+to+animal+testing+for+the+safety+assessment+of+cosmetics:+Eye+irritation.">A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Eye irritation.</a> Regulatory Toxicology and Pharmacology. 54 (2), 197-209 (2009).</li><li>Mathes, S. H., Ruffner, H., Graf-Hausner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+skin+models+in+drug+development.">The use of skin models in drug development.</a> Advanced Drug Delivery Reviews. 69-70, 81-102 (2014).</li><li>Abd, 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=Skin+models+for+the+testing+of+transdermal+drugs.">Skin models for the testing of transdermal drugs.</a> Clinical Pharmacology: Advances and Applications. 8, 163-176 (2016).</li><li>Flaten, G. 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=In+vitro+skin+models+as+a+tool+in+optimization+of+drug+formulation.">In vitro skin models as a tool in optimization of drug formulation.</a> European Journal of Pharmaceutical Sciences. 75, 10-24 (2015).</li><li>Avci, 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=Animal+models+of+skin+disease+for+drug+discovery.">Animal models of skin disease for drug discovery.</a> Expert Opinion on Drug Discovery. 8 (3), 331-355 (2014).</li><li>Mak, I. W., Evaniew, N., Ghert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+animal+models+and+clinical+trials+in+cancer+treatment.">Lost in translation: animal models and clinical trials in cancer treatment.</a> American Journal of Translational Research. 6 (2), 114-118 (2014).</li><li>Pronko, P. P., VanRompay, P. A., Zhang, Z., Nees, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronko+et+al.+Reply.">Pronko et al. Reply.</a> Physical Review Letters. 86 (7-12), 1387 (2001).</li><li>H.R.2858 - Humane Cosmetics Act. 114th Congress Available from: <a target="_blank" href="https://congress.gov/bill/114th-congress/house-bill/2858">https://congress.gov/bill/114th-congress/house-bill/2858</a> (2016)</li><li>. Global in-vitro toxicology testing market report: size, share &amp; trends analysis 2014-2015 Available from: <a target="_blank" href="https://www.prnewswire.com/news-releases/global-in-vitro-toxicology-testing-market-report-size-share--trends-analysis-2014-2025-300704958.html">https://www.prnewswire.com/news-releases/global-in-vitro-toxicology-testing-market-report-size-share--trends-analysis-2014-2025-300704958.html</a> (2018)</li><li>Zhang, Z., Michniak-Kohn, B. 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+engineered+human+skin+equivalents.">Tissue engineered human skin equivalents.</a> Pharmaceutics. 4 (1), 26-41 (2012).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+skin+corrosion:+reconstructed+human+epidermis+(RhE)+test+method.+Test+Guideline+No.431.">In vitro skin corrosion: reconstructed human epidermis (RhE) test method. Test Guideline No.431.</a> OECD Guideline for Testing of Chemicals. , (2019).</li><li>Almeida, A., Sarmento, B., Rodrigues, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+in+vitro+models+for+safety+and+toxicity+assessment+of+cosmetic+ingredients.">Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients.</a> International Journal of Pharmaceutics. 519 (1-2), 178-185 (2017).</li><li>vanden Broek, L. J., Bergers, L. I. J. C., Reijnders, C. M. A., Gibbs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+future+Prospectives+in+Skin-on-Chip+Development+with+Emphasis+on+the+use+of+Different+Cell+Types+and+Technical+Challenges.">Progress and future Prospectives in Skin-on-Chip Development with Emphasis on the use of Different Cell Types and Technical Challenges.</a> Stem Cell Reviews and Reports. 13 (3), 418-429 (2017).</li><li>Ata&#231;, B., 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=Skin+and+hair+on-a-chip:+In+vitro+skin+models+versus+ex+vivo+tissue+maintenance+with+dynamic+perfusion.">Skin and hair on-a-chip: In vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.</a> Lab on a Chip. 13 (18), 3555-3561 (2013).</li><li>Abaci, H. E., Gledhill, K., Guo, Z., Christiano, A. M., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pumpless+microfluidic+platform+for+drug+testing+on+human+skin+equivalents.">Pumpless microfluidic platform for drug testing on human skin equivalents.</a> Lab on a Chip. 15 (3), 882-888 (2015).</li><li>Wu, 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=Full-thickness+human+skin-on-chip+with+enhanced+epidermal+morphogenesis+and+barrier+function.">Full-thickness human skin-on-chip with enhanced epidermal morphogenesis and barrier function.</a> Materials Today. 21 (4), 326-340 (2017).</li><li>Materne, E. -. 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=The+multi-organ+chip+-+a+microfluidic+platform+for+long-term+multi-tissue+coculture.">The multi-organ chip - a microfluidic platform for long-term multi-tissue coculture.</a> Journal of Visualized Experiments: JoVE. (98), e52526 (2015).</li><li>Schimek, 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=Bioengineering+of+a+full-thickness+skin+equivalent+in+a+96-well+insert+format+for+substance+permeation+studies+and+organ-on-a-chip+applications.">Bioengineering of a full-thickness skin equivalent in a 96-well insert format for substance permeation studies and organ-on-a-chip applications.</a> Bioengineering. 5 (2), 43 (2018).</li><li>Alberti, 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=Multi-chamber+microfluidic+platform+for+high-precision+skin+permeation+testing.">Multi-chamber microfluidic platform for high-precision skin permeation testing.</a> Lab on a Chip. 17, 1625-1634 (2017).</li><li>Bhatia, S. N., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+organs-on-chips.">Microfluidic organs-on-chips.</a> Nature BIotechnology. 32 (8), 760-772 (2014).</li><li>Huh, D., Hamilton, G. A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+3D+cell+culture+to+organs-on-chips.">From 3D cell culture to organs-on-chips.</a> Trends in Cell Biology. 21 (12), 745-754 (2011).</li><li>Wufuer, 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=Skin-on-a-chip+model+simulating+inflammation,+edema+and+drug-based+treatment.">Skin-on-a-chip model simulating inflammation, edema and drug-based treatment.</a> Scientific Reports. 6, 37471 (2016).</li><li>Ramadana, Q., Ting, F. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+micro-physiological+immune-competent+model+of+the+human+skin.">In vitro micro-physiological immune-competent model of the human skin.</a> Lab on a Chip. 16, 1899-1908 (2016).</li><li>Kim, K., Jeon, H. M., Choi, K. C., Sung, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+effectiveness+of+Curcuma+longa+leaf+extract+on+a+skin+equivalent+using+a+pumpless+skin-on-a-chip+model.">Testing the effectiveness of Curcuma longa leaf extract on a skin equivalent using a pumpless skin-on-a-chip model.</a> International Journal of Molecular Sciences. 21 (11), 3898 (2020).</li><li>Halldorsson, S., Lucumi, E., G&#243;mez-Sj&#246;berg, R., Fleming, R. M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+challenges+of+microfluidic+cell+culture+in+polydimethylsiloxane+devices.">Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.</a> Biosensors and Bioelectronics. 63, 218-231 (2015).</li><li>Huh, D., Matthews, B. D., Mammoto, A., Montoya-Zavala, M., Hsin, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Huh, 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+human+disease+model+of+drug+toxicity+-+induced+pulmonary+edema+in+a+lung-on-a-chip+microdevice.">A human disease model of drug toxicity - induced pulmonary edema in a lung-on-a-chip microdevice.</a> Scientific Translational Medicine. 4 (159), (2012).</li><li>Beckwitt, C. H., 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=Liver+'+organ+on+a+chip+'.">Liver ' organ on a chip '.</a> Experimental Cell Research. 363 (1), 15-25 (2018).</li><li>Poceviciute, R., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-gut-microbiome+on+a+chip.">Human-gut-microbiome on a chip.</a> Nature Biomedical Engineering. 3 (7), 500-501 (2019).</li><li>Kanda, T., Sullivan, K. F., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-GFP+fusion+protein+enables+sensitive+analysis+of+chromosome+dynamics+in+living+mammalian+cells.">Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.</a> Current Biology. 8 (7), 377-385 (1998).</li><li>Esc&#225;mez, M. 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=Assessment+of+optimal+virus-mediated+growth+factor+gene+delivery+for+human+cutaneous+wound+healing+enhancement.">Assessment of optimal virus-mediated growth factor gene delivery for human cutaneous wound healing enhancement.</a> Journal of Investigative Dermatology. 128 (6), 1565-1575 (2008).</li><li>Llames, S. 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=Human+plasma+as+a+dermal+scaffold+for+the+generation+of+a+completely+autologous+bioengineered+skin.">Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin.</a> Transplantation. 77 (3), 350-355 (2004).</li><li>Llames, 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=Clinical+results+of+an+autologous+engineered+skin.">Clinical results of an autologous engineered skin.</a> Cell Tissue Bank. 7 (1), 47-53 (2006).</li><li>Cubo, N., Garcia, M., del Ca&#241;izo, J. F., Velasco, D., Jorcano, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+functional+human+skin:+production+and+in+vivo+analysis.">3D bioprinting of functional human skin: production and in vivo analysis.</a> Biofabrication. 9 (1), 015006 (2016).</li><li>Mori, N., Morimoto, Y., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+integrated+with+perfusable+vascular+channels+on+a+chip.">Skin integrated with perfusable vascular channels on a chip.</a> Biomaterials. 116, 48-56 (2017).</li><li>Kim, H. J., Li, H., Collins, J. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip.">Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (1), 7-15 (2016).</li><li>Shah, 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=A+microfluidics-based+in+vitro+model+of+the+gastrointestinal+human-microbe+interface.">A microfluidics-based in vitro model of the gastrointestinal human-microbe interface.</a> Nature Communications. 7, 11535 (2016).</li><li>Marx, U., 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=Human-on-a-chip'+developments:+A+translational+cuttingedge+alternative+to+systemic+safety+assessment+and+efficiency+evaluation+of+substances+in+laboratory+animals+and+man.">Human-on-a-chip' developments: A translational cuttingedge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man.</a> Alternatives to Laboratory Animals. 40 (5), 235-257 (2012).</li><li>Bein, 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=Microfluidic+organ-on-a-chip+models+of+human+intestine.">Microfluidic organ-on-a-chip models of human intestine.</a> Cellular and Molecular Gastroenterology and Hepatology. 5 (4), 659-668 (2018).</li><li>Bennet, D., Estlack, Z., Reid, T., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microengineered+human+corneal+epithelium-on-a-chip+for+eye+drops+mass+transport+evaluation.">A microengineered human corneal epithelium-on-a-chip for eye drops mass transport evaluation.</a> Lab on a Chip. 18, 1539-1551 (2018).</li><li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow.">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab on a chip. 12, 2165-2174 (2012).</li><li>Kim, H. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-on-a-chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation.">Gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation.</a> Integrative Biology. 5 (9), 1130-1140 (2013).</li><li>O'Neill, A. T., Monteiro-Riviere, N. A., Walker, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microfluidic+human+epidermal+keratinocyte+culture.">Characterization of microfluidic human epidermal keratinocyte culture.</a> Cytotechnology. 56 (3), 197-207 (2008).</li><li>Ren, K., Chen, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+materials+for+microfluidics+in+biology.">New materials for microfluidics in biology.</a> Current Opinion in Biotechnology. 25, 78-85 (2014).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amchafibrin</td>
			<td>Rottafarm</td>
			<td></td>
			<td>Tranexamic acid</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic</td>
			<td>Thermo Scientific HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plates</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape vynil</td>
			<td>ATP Adhesive Systems</td>
			<td></td>
			<td>GM 107CC, 12 &#181;m thick</td>
		</tr>
		<tr>
			<td>Edge plotter</td>
			<td>Brother</td>
			<td></td>
			<td>Scanncut CM900</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Extracted from human plasma</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Human dermal fibroblasts</td>
			<td>-</td>
			<td></td>
			<td>Primary. Gift from Dr. Marta Garc&#237;a</td>
		</tr>
		<tr>
			<td>H2B-GFP-HaCaT cell line</td>
			<td>ATCC</td>
			<td></td>
			<td>Immortalized keratinocytes. Gift from Dr. Marta Garc&#237;a</td>
		</tr>
		<tr>
			<td>Live/dead kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>Merk TM</td>
			<td></td>
			<td>5 &#181;m pore size</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Sylgard 184</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Terumo</td>
			<td></td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>10 NIH/vial</td>
		</tr>
		<tr>
			<td>Transparent adhesive vinyl</td>
			<td>Mactac</td>
			<td></td>
			<td>JT 8500 CG-RT, 95 &#181;m thick</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>IDEX</td>
			<td></td>
			<td>Teflon, 1/16&#8221; OD, 0.020&#8221; ID</td>
		</tr>
	</tbody>
</table>

generation of a simplified three-dimensional skin-on-a-chip model in a micromachined microfluidic platform

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。

设计的芯片由两个流体室组成，由一个5μm孔径的PC膜隔开，该膜允许细胞通过允许促进生长的分子从下腔室通过。上腔室容纳组织构建体，在这种情况下，在含有hFBs的纤维蛋白水凝胶上放置单层hKCs。
通道的高度由添加到每个通道的粘合片数量决定。下腔室由4层（380μm）和10个单面胶带层和双面带层（962μm）组成。芯片的尺寸为4 cm x 2 cm，这增强了其操作性。粘性乙烯基板为?...

Watch this Scientific Journal Video about Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform at JoVE.com

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

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。

在微加工微流体平台中生成简化的三维芯片皮肤模型

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

这是一种新颖的、无光刻的协议，用于使用微加工的粘性乙烯基层和用于真皮室生成的平行流动方法对3D芯片皮肤进行建模。微加工乙烯基在制造过程中提供了更简单的工艺，在器件的布局中提供了多功能性，克服了PDMS和传统光刻方法的一些局限性。这种多层片上皮肤可用于药物和化妆品测试方法，因为它允许以较低的成本进行高通量筛选，并可用于对多种疾病进行建模。首先使用Brother Canvas Workspace软件设计微流控芯片。创建一个 30 x 30 厘米的工作空间，并在将其存储在 svg 文件中之前，用芯片不同层的设计模式填充它。剪切 30 x 30 厘米的乙烯基板。将其粘贴到低粘性粘合剂垫上，并根据需要消除所有气泡。使用 USB 驱动器，将 svg 文件上传到边缘绘图仪，并将切割参数设置为零级切割压力、三级切割刀片和一级切割速度。设置参数后，将带有乙烯基的粘合垫放入绘图仪中，然后开始在95微米厚的乙烯基上切割通道图案的过程。使用对准器组装整个设备，以正确调整通道、入口和出口。用相应的微型机器设计堆积乙烯基层，用于组装下部通道，保持底层的盖带以避免粘在对准器上。将聚碳酸酯多孔膜切割并放置在下通道的顶部，而不覆盖入口。添加10个具有上腔室设计的乙烯基层，并在顶部粘贴双面胶带最后一层。从对准器中取出芯片，将其粘贴在载玻片上。将两毫米厚的 PDMS 纸放在双面胶带乙烯基层的顶部。为确保芯片完全防水，请在芯片顶部放置一个砝码过夜。第二天，用70%乙醇冲洗芯片五分钟，然后用蒸馏水清洗芯片，对芯片进行灭菌。将泵一和泵二分别连接到上腔入口一和上腔入口两。将泵三连接到下腔室入口，然后将上腔和下腔出口连接到废液桶。使用 PTFE 管和 18 号不锈钢连接器将注射器连接到每个入口。泵PBS用泵三通过下腔入口，每分钟50微升，用泵两通过上腔进气口，每分钟100微升。然后，将纤维蛋白原预凝胶加载注射器，立即将其放入泵中，并以每分钟200微升的速度运行。一旦预凝胶从上腔室出口出来，停止泵一泵和泵二泵，然后在不取下管子的情况下，离开芯片以允许在37摄氏度下凝胶化10分钟。凝胶化后，用盖子堵住上腔入口，并将培养基泵入上腔入口2，泵三，每小时50微升过夜。在生成真皮隔室24小时后，检查人原代成纤维细胞是否在组装体中扩散，然后通过上通道入口2以每分钟40微升的速度引入5次10至6 HKCs，每毫升1分钟。对于细胞附着，将芯片在37摄氏度下在湿度饱和的培养箱中放置过夜。开始用泵三泵泵新鲜培养基，仅通过下腔室入口，每分钟50微升。在代表性分析中，显示沿组件上腔的水凝胶高度。牺牲PBS和预凝胶的平均高度为550微米，在100和200微升/分钟的流速下，凝胶的功能是最佳的。未能通过下通道冲洗PBS导致凝胶的理论高度与被测高度之间的差异，相差40%从平行流方案启动24小时后，发现人类原发成纤维细胞成功地在组装上腔的纤维蛋白凝胶中扩散， 之后，绿色荧光蛋白表达HKCs可以有效地接种在水凝胶的顶部。去除管子而不保持系统关闭过夜以允许HKCS沉淀导致细胞不均匀，汇合的单层细胞。微流控芯片中未分化皮肤模型的3D共聚焦分析清楚地显示了将顶部的HKCs与嵌入底部的成纤维细胞分开的水凝胶表面。生成的皮肤可以暴露于空气/液体界面以获得成熟和分化的表皮，并使用几种真皮和表皮标志物进一步表征。该技术允许更快，更便宜的设备制造，并使用微流体生成多层皮肤，为药物和化妆品测试提供更现实的方法。

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...