Name: use of a high-throughput in vitro microfluidic system to develop oral multi-species biofilms
Uploaded: 2014-12-01T15:00:00
Description: Watch this Scientific Journal Video about Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms at JoVE.com

笔者在制定生物膜增长协议和约翰·巴蒂斯塔（流动现状，旧金山，加利福尼亚州）的关于与该Bioflux系统的技术问题咨询感谢威廉·南斯（密歇根大学）寻求帮助。密歇根大学（R21DE018820至AHR NIH）和启动资金，AHR这项工作是由美国国立卫生研究院的支持

这里所描述的唾液收集协议是由美国密歇根机构审查委员会的大学人体试验审查。 注：对于机构的评论对这类人的主体工作，事先安排和权限应该由主办机构可以囊括。特别是，根据该机构，IRB或伦理委员会批准可能需要寻求批准，从人类志愿者的唾液采集前可以继续进行。为了帮助准备申请，一个有用的NIH算法/图可以在这里找到： <a href="http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDe...

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此方法本文强调需要设置的基本步骤和方式运行一个微流体系统，以允许对来自于合并的人唾液和在过滤除菌的25％合并的人唾液生长口服多品种生物膜发展。方法来表征所述生物膜中给出，但应当记住，这些描述的方法进行修改和附加的技术，如，例如，污渍或标签可以引入。作为例子的问题，人们可以设想使用标记的抗体或引入的荧光应变来可视化和检查某些物种的口服多品种生物膜内的空?...

<html>生物膜是细菌架构复杂的社区聚集在表面1。这些社区通常包含许多物种，与彼此生物膜2内进行交互。口服生物膜，视觉上最显着的是牙菌斑，是在人类中一个长期存在的问题和其不受控制的发展成果在分类学上不同的多种类的社区3的生成。这些不同的社区的组分的细菌可高达1000倍于抗微生物剂比它们的自由浮动的（浮游）对口4-6更有抵抗力。如果不能把这些口腔生物膜的社区，这可能会导致龋齿和牙周病，导致了显著的公共卫生负担：超过500万人次，在美国每年的牙科诊所，以及约108十亿治疗或预防牙周疾病和龋齿7。 内容“ - &gt;” 虽然许多微生物学家崇尚自然条件下学习微生物的行为，很少有人这样做。这是因为他们的士气，克服困难不断的有吸引力的易用性实验室培养的工作 。“-Smith 8 大伤元气 。 目前，口腔生物膜研究使用各种在体内和体外的方法进行的，每个都有自己的优点和缺点9,10。 在体外方法经常使用的模型生物膜系统，是比较容易建立，但可能缺乏临床/真实世界的相关性10,11。 体内方法通常依赖于可能重现人口腔环境的某些方面，但再次从限制遭受由于动物间的差异在解剖学，生理学，微生物学和免疫学和人类12动物模型系统， 13。应当指出的是口服生物膜也可在人类志愿者的口内的支架保持搪瓷面开发的，但这种方法是目前比较昂贵和劳动密集14,15。最终，新药或技术，以改善口腔卫生保健在对照临床试验条件11人进行测试。目前，一个经常使用的手法进行识别和评估新的口腔护理剂，是先进行实验室研究，以识别潜在的功效，然后执行动物研究和“实地测试”雇用医生评估技术9的成功， 16,17。不幸的是，实验室的研究往往依赖于占有相当大的足迹模型系统，在技术上具有挑战性的使用，而且往往包含简化的一个或最多几个种类社区获得潜在的现实世界的意思10,18。由于牙菌斑生物膜包含一个并发症多种类和形式前流唾液环境，开发含有一个生物膜或几个物种中人工介质不大可能产生其行为以类似的方式中的那些真实世界的场景10,19社区。为了解决这个时间，成本，培训要求，和实验室模型生物膜系统的差的代表性相比真实世界环境，我们最近开发的高通量和环境锗生物膜系统20（ 图1）。该系统得益于采用无细胞汇集人的唾液（中心）作为培养基和未处理的合并的人细菌含细胞唾液（CCS）作为接种物。与众不同的是，该系统还结合了微流体技术，激光共聚焦扫描显微镜和文化无关的细菌多样性的分析技术。因此，该模型系统是环保锗（使用唾液作为接种到在37种多品种生物膜 °下在过滤灭菌的流动唾液）和口腔生物膜中含有丰度代表那些在早期龈上菌斑发现20种（包括链球菌 ， 淋球菌，Veillonella和卟啉类）。 当考虑到这项工作介绍了利用新开发的模型系统，必须要特别注意给予激光共聚焦扫描显微镜（CLSM）微流控技术的融合，文化无关的多样性分析技术。这些技术我们研究小组的联合是故意的，不仅增加了高吞吐能力，以新开发的模型系统，也可以让问题要问，可能不容易与其他系统解决之前。首先，CLSM具有比传统的显微镜具有明显的优势，因为它允许生物膜的三维分析。往往不受重视，这是极其重要的，因为生物膜是异构的智慧ħ对于物种组成和空间位置，以及在生理条件被强加在生物膜6,21内的不同空间位置。在音乐会三维渲染软件和图像分析软件，生物膜结构，部件种类之间的空间关系，以及抗微生物杀灭程度可以分析22-24。这样的能力是不可能使用标准透射光或荧光显微镜。接着，微流体已经获得特别关注在微生物学领域，因为它能使小心控制的条件（流量，温度，pH 等 ）下生物膜的研究，只需要小体积的液体25-27。作为比较点，生长在人唾液的口腔生物膜的流动细胞模型系统（即可以说是考虑了支柱模型对许多口服生物膜研究的系统）20小时之内，在一个类似的流速和剪切作为实现在微流体系统至少需要200毫升，如在微流器件28-31反对800微升。因此，微流体模型生物膜系统，使数量受限物质的特定条件下的研究。最后，焦磷酸​​测序技术已经在过去十年中优化以只需要少量的材料制成，以执行一个社区的分析和足够通用的，以控制测序深度以得到甚至稀有生物膜物种的身份。利用这种技术，如细 ​​菌标记编码FLX扩增子焦磷酸测序（bTEFAP），已经允许有关生物膜的生态相关问题加以解决32,33。这样的问题，当浸透焦磷酸测序是因为时间和创建的质粒库并从中获得数据33,34所需的复杂的技术和分析步骤所需要的成本无法在过去的困难。当然，与文化无关的AP具有很大的优势proaches，如焦磷酸测序，是常规实验室介质（ 即活的但非可培养的物种）中不能生长在隔离的细菌种类可以生长，并确定了该模型系统内以及它们的相对丰度在社区量化35,36 。加的角度看，早在1963年，在后期西格蒙德Socransky估计中的细菌的材料从人口腔齿龈裂缝中分离的约50％的不能使用实验室生长条件37培养。 此方法本文的目的是描述该方法下，发展中的市售微流体（Bioflux）系统口服多品种生物膜：（ⅰ）代表的条件的人口腔的和（ii）带有一个物种组成和数量那媲美龈上菌斑。此外，同时使用免费软件和商业软件，我们强调如何基本生物膜建筑措施可以从CLSM数据导出，侧重于方法来量化生物膜生物量，粗糙度和可行性（基于活/死染色）。最后，通过bTEFAP收获多样性分析生物膜材料所需的步骤如下所述。

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use of a high-throughput in vitro microfluidic system to develop oral multi-species biofilms

很少有高通量的体外系统，促进包含在体内口服生物膜内常用检测无数种多品种生物膜的发展。另外，一个系统，它使用代替人工介质的自然人类唾液作为营养源，是特别理想的，以支持对模仿体内社区蜂窝和生物膜特定的属性的表达。我们描述了一种用于多品种口服生物膜是可比，相对于组合物物种的发展，对龈上菌斑，类似于人类口腔条件下进行。具体而言，该方法本文将描述如何市售微流体系统可适于促进衍生自多品种口服生物膜的发展，并汇集唾液内生长。此外，该系统是如何的描述，可以结合使用一个confoca升激光扫描显微镜生成将提交3-D生物膜重建的建筑和可行性分析。定在微流体系统（包括链球菌属 ， 奈瑟氏球菌，Veillonella，Gemella酒店 ，和卟啉 ）生物膜内生长了微生物的广泛多样性，协议也将提交描述如何收获生物膜细胞用于进一步传代培养或DNA的提取和分析。无论是微流控系统的生物膜和国家的最先进的当前数据分析的范围将得到解决。最终，可以设想，本文将提供一个基线技术，这将提高口腔生物膜研究和有助于在可与微流体平台被整合额外的技术的开发。

生物膜的3D渲染 代表性的结果示于图3中 。在IMARIS软件的有用工具是考察所收集的生物膜栈的每个切片，并组合它们以创建三维重建的选项。此外，可以加入人工遮蔽效果，以帮助在视觉上解释的三维结构。呈现的生物膜可定向在不同的放大倍数，探讨生物膜或微生物菌落结构的任何方向。示于图中的图像。图3示出一...

Watch this Scientific Journal Video about Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms at JoVE.com

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

此方法本文的目的是描述了使用微流体系统的为包含通常在人类龈上菌斑鉴定物种多品种生物膜的发展。方法描述生物膜结构，生物膜的可行性，以及一种方法来收获生物膜文化依赖或培养独立分析突出显示。

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

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Research

JoVE Journal

Bioengineering

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

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High-throughput Protein Expression Generator Using a Microfluidic Platform

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

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

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Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

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To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

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Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

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Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

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Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...