Name: use of a high-throughput in vitro microfluidic system to develop oral multi-species biofilms
Uploaded: 2014-12-01T15:00:00.000+00:00
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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/PrivateInfoOrBioSpecimensDecisionChart.pdf">http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDecisionChart.pdf</a> 1.准备汇集唾液用作环境锗生长培养基<ol><li>招聘唾液捐赠&gt; 5个人。不要采取任何身份信息，确保无individu捐赠前，ALS将捐出口水，如果他们生病，或已经在近3个月采取口服抗生素，或已经消耗的食物或液体，除了水，在之前的2小时。 </li><li>在50毫升的塑料管收集唾液。池收集唾液在塑料烧杯中，保持它在冰上。不使用玻璃作为在唾液中的聚合物会附着在内部的玻璃表面上。 </li><li>从100X库存添加二硫苏糖醇（DTT）至2.5mM的终浓度。 （股票被冻结时的一次性使用分装-20 ℃）。搅拌在冰上塑料烧杯中10分钟。 </li><li>为了去除颗粒物质，离心机汇集唾液30分钟，在17500×g下。 </li><li>冲淡唾液与3倍体积的dh 2 O，给予四分之一集中口水。 </li><li>过滤消毒使用0.22微米的聚醚砜（PES），低蛋白结合过滤口水。使用过滤器具有大的表面积，或者使用几个小的区域的过滤器。保持唾液的冰上塑料容器而过滤。 </li><li>冻结汇集唾液在-80 &#160; ℃，在50毫升的塑料管，直到需要滋长细菌。每个塑料管是只使用一次，并应含有不超过35 ml为每个微流体以及最多容纳1.2毫升（1.2毫升×24个孔=28.8毫升）和空间需要在每一个管作为冷冻过程中的唾液展开。 </li><li>在使用时，在室温下解冻汇集唾液。一旦解冻，过滤消毒一次（0.22微米聚醚，低蛋白结合过滤器，以除去任何沉淀物）。 </li></ol> 2.准备汇集唾液用作接种<ol><li>招聘唾液捐赠&gt; 5个人。不要采取任何身份信息，确保没有人会捐出口水，如果他们生病，采取口服抗生素，在过去3个月，已经消耗的食物或液体，除了水，在之前的2小时献血前。收集SAmples在30分钟内。 </li><li>收集唾液在50ml塑料管中，在室温下，集中收集唾液在塑料烧杯在室温下。 </li><li>汇集稀释唾液蒸压一般试剂级甘油产生的股票有25％甘油的最终比例，有75％含有细胞的唾液[CCS]。 </li><li>直到需要在3ml一次性使用的等分试样冷冻唾液在-80℃。 </li><li>当需要接种的微流体系统，将等分试样在协议中的步骤3中描述，在下面被吸移到微流体系统之前在室温下解冻，轻轻搅动在涡旋5秒。 </li></ol> 3.生长口服多品种生物膜<ol><li> CFS预处理 <ol><li>第一层的Bioflux微流体通道CFS。加入100微升CFS的每个出口处好。使用Bioflux控制软件，选择频道的"使用说明书"，并设置流动"B1-B24"塔t为被使用。 </li><li>接下来，设置剪切至1.0达因/平方厘米，并开始流动，在室温下2分钟，以确保CFS的整个通道均匀分布。确保有流体在每个入口通道，以验证CFS通过各种渠道流入均匀。 </li><li>孵育板在室温下20分钟。 </li><li>除去CFS /的预处理溶液残留在所述出口孔中，并转移到所述入口孔。 100微升这种总体积将有助于对抗压力平衡被施加到接种物从出口井。 </li></ol></li><li> 接种 <ol><li>每个插座均匀，加入100微升CCS接种。放置在微流体片上热板（温度设定为37℃）时，控制软件内选择手动，并从出口孔设置流动到入口孔（ 即 ，后退）在1.0达因/平方厘米，精确6秒。 </li><li>孵育微流体板在37℃下40分钟，以允许用于初始粘附和细菌在接种物生长的影响。 </li></ol></li><li> 隔夜增长 <ol><li>对于所使用的接种途径，从每个出口孔吸出废接种物。加起来CFS 1毫升总量到每个入口井（可在现有CFS之上完成）。 </li><li>孵育微流体板在37℃下，选择手动，并设置程序为0.2达因/平方厘米运行20小时。 </li></ol></li><li> 预洗污点 <ol><li>抽吸所有从入口和出口孔的流体，并添加100微升PBS（pH7.4）中，以各所述入口孔。流20分钟，以0.2达因/厘米2。 </li></ol></li><li> 染色混合另外 <ol><li>对于细胞活力染色每个通道使100μl的染色混合物的被染色。具体地，添加3微升SYTO 9和3μl的每毫升PBS中使用商业细胞活力染色试剂盒的碘化丙锭的诸如活/死。这会产生染色混合物，含10μMSYTO 9和60μM碘化。 </li><li>从入口孔吸出剩余的PBS中，然后添加100微升的细胞活力染色混合物与每个入口井。设置为流动0.2达因/厘米2，并运行从入口溶液出口，在室温下45分钟。 </li></ol></li><li> 后染色洗 <ol><li>吸出剩余的污点在每个入口孔和添加100微升PBS中，以每个入口井。设置为流动0.2达因/平方厘米，并运行从入口PBS溶液至出口20分钟，在室温下，以除去任何过量的污点。 </li></ol></li></ol> 4.图像采集，3D渲染和图像分析<ol><li>使用倒置共聚焦激光扫描显微镜（CLSM）从微流体系统收集生物膜的图像数据。确保CLSM是能够承受Bioflux板，其可达到150克的重量。 注:鉴于角钱微流体通道，需要灵敏度辨别生物膜结构，并且要求nsions以检测不同强度的荧光信号，具有40X 1.25 NA物镜或具有类似的光学质量，倍率及数值孔径适应CLSM。 </li><li>规范排放捕获门与增益和偏移测量保持恒定。确保激光功率不超过25％，因为这可能会导致光漂白。 </li><li> 从 L EICA 我法师˚FILE类型（LIF），以OME（O笔M icroscopyËnvironment）文件类型转换CLSM文件。这允许更大的易于使用的软件程序之间的访问和兼容性。使用市售软件如，IMARIS将文件转换为这种类型。 </li></ol> 3D渲染的图片/图 <ol start="4"><li>一旦转化为OME格式，请使用市售软件如IMARIS渲染图像在3D。使用"Easy3D"和"超越"选项的组合执行此。 注意:注意背景信号和阈值应作出。在IMARIS直方图函数可用于获得采集范围和本应保持恒定之间的图像被分析。 </li><li>保存使用" 快照 "功能，图像和保存文件类型之前周密考虑的图像分辨率。 </li><li>组装成图像用图像编辑软件的数字，如CorelDraw中或Adobe Illustrator中。 </li></ol> 对于图表3D图像分析 <ol start="7"><li>使用免费提供的ImageJ 23和COMSTAT / COMSTAT2 38图像分析。从http://rsb.info.nih.gov/ij/和http://comstat.dk/，下载包分别。已经安装了最新的Java更新。 注:软件平台的JAVA将需要安装PRIOr以使用该图像分析软件。 <ol><li>使用ImageJ的软件COMSTAT2插件导入OME文件中的"hyperstack"模式，并针对每一个生物膜形象" 分割通道 "。 </li><li>单独分析两个通道（红/碘化碘化/死存在通道0，绿色/ SYTO-9 / LIVE是通道1）使用ImageJ的" 直方图"功能。此函数列出的8位OME文件像素（显示为"计数"），在每种颜色的强度从0到255（显示为"值"）的总数，其中0无信号（背景）作为象素和255的存在像素完整的信号饱和。 </li><li>数据导出到电子表格程序，并通过相应的信号强度加权的各像素的标准化所有信号值。这是通过由该信号强度的数值的8位值（0-255）乘以在给定信号强度的总数进行。 </li><li>总结所有的加权值，0-255，两个通道的捕捉每一个生物膜的形象。取加权值的总和为两个通道，以确定从任一通道中的％的总的信号。这样做是为了确定红色和绿色的百分比信号（ 即 ％的"死"和百分比"LIVE"每处理）的相对比率或百分比。 </li><li>执行统计分析，例如，使用双尾学生t检验修改后用于方差不等。 P &lt;0.05的值被认为是显著和P &lt;0.01的值被认为是高度显著。 </li></ol></li></ol> 5.收获生物膜细胞培养独立分析<ol start="1"><li>从入口和出口的油井删除所有花费和未使用的口水。用1毫升无菌蒸馏水洗涤三次洗涤的孔中。 </li><li>加入100微升无菌蒸馏水到入口孔，并使用软件控制的接口，通过无菌蒸馏水前进通过通道在&gt; 8.0达因/厘米2（流速&gt; 745微升/小时，抗剪800秒-1）为至少5分钟。重复在相反的方向，至少5分钟，然后重复正向和反向洗涤步骤的过程。 </li><li>检查通过光或通过荧光显微镜（如果细胞染色/标记），以便进行彻底生物膜去除（ 图2）。收集100微升细胞悬液，并储存在-80℃下培养独立分析。可以通过使用使用拔头筹等人描述的方法细菌标记编码FLX扩增子焦磷酸测序（bTEFAP）来实现群落组成的具有成本效益的分析。20 </li></ol>

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Total microscopic and viable counts and counts of specific organisms.</a> Archives of oral biology. 8, 275-280 (1963).</li><li>Heydorn, 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=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (10), 2395-2407 (2000).</li><li>Nobbs, A. H., Lamont, R. J., Jenkinson, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+adherence+and+colonization.+Microbiology+and+molecular+biology+reviews).">Streptococcus adherence and colonization. Microbiology and molecular biology reviews).</a> MMBR. 73 (3), 407-450 (2009).</li><li>Hope, C. K., Clements, D., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+spatial+distribution+of+viable+and+nonviable+bacteria+in+hydrated+microcosm+dental+plaques+by+viability+profiling.">Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling.</a> Journal of applied microbiology. 93 (3), 448-455 (2002).</li><li>Adams, 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=Development+of+a+laboratory+model+to+assess+the+removal+of+biofilm+from+interproximal+spaces+by+powered+tooth+brushing.">Development of a laboratory model to assess the removal of biofilm from interproximal spaces by powered tooth brushing.</a> American journal of dentistry Spec No 12B-17B. 15, 12-17 (2002).</li><li>Ledder, R. G., McBain, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+comparison+of+dentifrice+formulations+in+three+distinct+oral+microbiotas.">An in vitro comparison of dentifrice formulations in three distinct oral microbiotas.</a> Archives of oral biology. 57 (2), 139-147 (2012).</li></ol>

A. H. R. and N.S.J. have received research awards from a variety of sources such as the National Institutes of Health (NIDCR), Colgate-Palmolive (Piscataway, NJ) and the Society for Applied Microbiology to fund research studies in their lab over the past five years. All other authors: none to declare.

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。

<table><tbody><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Falcon 15 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Dithiothreitol (White Crystals or Powder/Electrophoresis), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Sorval ultracentrifuge&amp;nbsp; (SS-34 compatible)</td><td>Thermoscientific</td><td>Unit-dependent</td><td></td></tr><tr><td>Thermo Scientific SS-34 Rotor&amp;nbsp;</td><td>Thermoscientific</td><td>28-020</td><td></td></tr><tr><td>Thermo Scientific Type 1 Reagent Grade Deionized Water</td><td>Thermo&amp;nbsp; Scientific Inc</td><td>23-290-065</td><td></td></tr><tr><td>Nalgene Rapid-Flow Filter Units and Bottle Top Filters, PES Membrane, Sterile.</td><td>VWR</td><td>73520-986&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific Inc</td><td>NC0542269</td><td></td></tr><tr><td>BioFlux microfluidic system</td><td>Fluxion</td><td>Bioflux 200 system</td><td></td></tr><tr><td>Bioflux 24-channel plate</td><td>Fluxion</td><td>910-0004</td><td></td></tr><tr><td>PBS (Gibco)</td><td>Thermo Fisher Scientific Inc</td><td>10010023</td><td></td></tr><tr><td>LIVE/DEAD stain (Invitrogen)&amp;nbsp;</td><td>Invitrogen</td><td>L7012</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Lecia</td><td>SPE or eqivalent system</td><td></td></tr><tr><td>Epifluorescence Microscope</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Pyrosequencing facilities</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Decon SaniHol 70 Ethanol Solution</td><td>Fisher Scientific</td><td>04-355-122</td><td></td></tr><tr><td>Ultra Low Temperature Freezer -80&amp;nbsp;&amp;deg;C</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Tips (20, 200, and 1,000&amp;nbsp;&amp;#956;l)</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td>Single Channel Variable Volume Pipettors (20, 200, 1,000 &amp;#956;l)</td><td>Multiple choices</td><td>Multiple choices</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>Bioflux dedicated software</td><td>Bioflux</td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Leica SPE</td><td>Leica</td><td></td><td></td></tr><tr><td>ImageJ</td><td>Freeware (http://imagej.nih.gov/ij/)</td><td></td><td></td></tr><tr><td>COMSTAT/COMSTAT 2</td><td>Freeware (http://www.comstat.dk/)</td><td></td><td></td></tr></tbody></table>

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

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

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

13.4K 次观看.  The University of Michigan. 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。 

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

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

科研

免疫与感染

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

生物工程

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

生物化学

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

研究多孔介质中生物堵塞的微流体平台

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

环境科学

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

使用高通量的

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