Name: applying advanced in vitro culturing technology to study the human gut microbiota
Uploaded: 2019-02-15T14:45:00
Description: Watch this Scientific Journal Video about Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota at JoVE.com

奥黛丽·托马斯-加赫林女士因其 gcms 的工作而受到表彰。我们还要感谢马西莫·马尔佐拉蒂编辑的手稿。

1. 材料和制剂
请注意:定义的介质是作为粉末购买的 (见材料表)。g/l 中定义介质的组成如下: arabinogalactan (1.2)、pectin (2.0)、xylan (0.5)、葡萄糖 (0.4)、酵母提取物 (3.0)、特殊肽 (1.0)、粘液素 (2.0)、l-半胱氨酸-hcl (0.2)。
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准备定义的介质
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<li>用双蒸馏化、去离子水填充4升的烧瓶。</li>
		<li>在水中加入29.2 克的中粉, 混合装置完全均匀?...

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建立了体外培养系统, 研究大肠肠道微生物群。他们使用的设备设计, 以模拟胃肠道的生理条件, 促进一个成熟的肠道微生物群落的生长, 为结肠33的每个区域。虽然这个概念是合乎逻辑和可理解的, 但研究肠道微生物区系的体外培养系统的实际运行需要精确和了解所需的内容和预期产生可靠结果的情况。
体外肠道微生物区系实验所需的元素是, 群落在接种后必...

肠道微生物群是一个微生物群落, 存在于人类胃肠道 (git) 中。 这个群落在结肠中达到最大浓度, 估计可容纳 10 13-1014 种细菌, 来自500-1000 种, 与结肠环境 1,2共生。肠道微生物群的组成和功能沿 git 发生了空间变化, 形成了区域特定的群落, 最多样化的是远端 2、3、4、5。对于每个解剖区域, 不同的微生物群落居住在腔内和粘膜衬里 6。当底物穿过腔室 7时 7时7时, 腔内社区可以更直接地获得营养物质。尽管如此, 一些细菌优先居住在粘液层, 利用结肠细胞产生的粘液作为能量来源1,5,8。腔内和黏膜相微环境的差异导致了分化和特定期群落的发育。这些群落共同提供代谢功能, 如营养代谢和维生素的产生, 以及免疫功能, 如防止人类病原体的殖民1,3,9. 肠道微生物区系在与人结肠细胞的共轭作用下也起作用3。
作为人类 git 的重要组成部分, 众所周知, 肠道微生物群对宿主健康和疾病状况的贡献3、9、10、11、12 也就不足为奇了。肠道微生物群的变化与多种人类疾病有关, 包括肠道疾病 (ibd) 和肠道综合征 (ibs) 等 git 疾病, 以及肥胖、循环系统疾病和自闭症等其他疾病3 个,9,10,11,12. 肠道微生物群产生的代谢物具有全球效应, 到达远离肠道的地方12、13.例如, 肠道-大脑轴与焦虑和抑郁等精神障碍有关.因此, 研究肠道微生物群对多个研究领域都很重要, 适用于许多疾病, 即使是那些与 git 并不经常相关的疾病。
虽然人们普遍承认, 研究肠道微生物群很重要, 但这是一项复杂的工作。有多种动物模型, 从斑马鱼、老鼠和老鼠等小动物, 到猴子和猪 15-19等较大的动物。然而, 这些动物在人体肠道微生物区系方面的应用并不简单, 因为这些动物有一个独特的细菌群落, 这种群落是在环境和饮食的基础上进化而来的, 它们在解剖上与人类不同,21. 使用人的主体消除了相关性问题, 但又带来了另一组挑战。人类研究是昂贵的, 耗时的, 并在道德上受到限制11。此外, 混淆因素影响肠道微生物群在人类研究中, 包括年龄或发育阶段, 环境, 饮食, 药物, 和遗传因素2,4, 22。 对可以在人类身上检测的东西, 以及可以在什么时候采集哪些类型的样本也有限制 4。
使用体内系统研究肠道微生物群的一个关键缺点是哺乳动物成分的存在。肠道微生物群和人体细胞相互作用, 在体内环境中, 不可能区分这两者。肠道微生物群产生的代谢物由结肠细胞吸收, 因此无法精确计算测量结果。因此, 任何机械研究都必须限于终点测量 11.体内研究的另一个主要缺点是无法纵向从 git 23 的不同区域采集样本.这不允许评估随着时间的推移结肠微环境中可能发生的变化 12. 包括人类研究在内的许多体内研究都依靠粪便样本分析来检测肠道微生物群的变化。虽然这是提供信息, 但它没有提供整个 git 肠道微生物群的数据, 也没有区分腔和黏膜群落 5、6、7、8。
对于肠道微生物群, 需要应用体外方法来研究细菌群落的动态, 而不受哺乳动物成分的干扰。使用体外方法可以严格控制环境条件 10, 同时测试多个参数, 并能够进行纵向采样, 并在大容量11。由于体外方法使用的是机械设备, 而不是主机, 因此不需要考虑年龄、环境、饮食或遗传背景。这些系统可以用来测试整个肠道微生物群群落, 只能测试选定的生物, 甚至是单一的菌株。重要的是, 体外结果是可重现的, 但仍保留了与体内研究11,22相当的多样性水平。
根据所涉及的假设和预期的结果, 可以通过多种方式进行体外研究。 它们可以利用单血管系统和简单的方法, 例如用粪便均质24小时孵化样品, 或在 24-48 25期间进行单批培养。它们还可以使用单血管系统和更复杂的方法来完成, 例如使用恒温器系统来产生稳定的肠道微生物群落11。然而, 使用单个反应器可以过度简化微生物群12,因为它只代表结肠的一个部分, 即使结肠是由上升、横向和下降区域组成的。
为了研究在结肠不同区域 (上升、横向和下降区域) 发育的肠道微生物群群落, 可以采用复杂的多阶段系统。在这些系统中, 建立了多个血管来模仿结肠的不同区域, 因此上升、横向和下降区域的肠道微生物群是独立培养的。这些容器是连接的, 使用泵按顺序移动基板, 从上升到横向到下降的结肠区域, 模仿营养物质通过 git 的流动。
本研究的目的是展示如何利用5个阶段的体外培养系统 (见材料表) 来培育肠道微生物群群落, 并展示稳定和组成方面的群落动态。在这个系统中, 一个血管代表胃, 一个代表小肠。冒号分为三个区域 (上升、横向、下降), 每个区域为26个区域.在这一实验设置中, 两个完整的系统并行运行, 第1单元包含粘液载体, 以表示黏膜表面, 第2单元不含粘液载体。利用 16s rrna 基因测序和 scfa 分析, 对每个区域在腔和黏膜阶段发育的群落进行了比较, 并对一段时间内的粪便接种进行了比较。所提供的结果证明了社区的类型, 无论是在组成和功能方面, 都可以从这种类型的体外系统中产生。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TWINSHIME</td>
			<td>Prodigest</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>defined medium (Adult M-SHIME growth medium with starch)</td>
			<td>Prodigest</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex tubing</td>
			<td>cole Parmer</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Urine Drainage bag</td>
			<td>Bard</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Labsorb</td>
			<td>Sigma-Aldrich</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fecal sample</td>
			<td>Openbiome</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Becton Dickson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Defined medium</td>
			<td>Prodigest</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxgall Bile</td>
			<td>Becton Dickson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancreatin</td>
			<td>Sigma-Aldrich</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass ware</td>
			<td>Ace Glass</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine mucin</td>
			<td>Sigma-Aldrich</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Sigma-Aldrich</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization pouches</td>
			<td>VWR</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>BeadBug</td>
			<td>Benchmark Scientific</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple-Pure High Impact Zirconium 0.1mm Bead beater tube</td>
			<td>Benchmark Scientific</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free, DNAse free, sterile water</td>
			<td>Roche</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Shimadzu QP2010 Ultra GC/MS</td>
			<td>Shimadzu</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilwax-DA column, 30m, 0.25mm ID, 0.25&#181;m</td>
			<td>Restek</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic mucin carriers</td>
			<td>Prodigest</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying advanced in vitro culturing technology to study the human gut microbiota

人体肠道微生物群在人类健康和疾病中都发挥着至关重要的作用。利用体内模型研究肠道微生物区系是困难的, 因为它的复杂性, 以及它与哺乳动物成分的不同联系。该协议的目的是在体外培养肠道微生物群, 从而能够研究肠道微生物群动态, 而不必考虑哺乳动物环境的贡献。利用体外培养技术, 模拟了胃肠道的生理条件, 包括 ph 值、温度、厌氧菌和转运时间等参数。通过添加黏液涂层载体, 创建黏膜相, 并添加进一步的维度, 模拟结肠的肠道表面。通过接种人体粪便材料, 引入肠道微生物群。在接种这种复杂的细菌混合物时, 特定的微生物在不同的纵向 (上升, 横向和下降冒号) 和横向 (腔和粘膜) 环境的体外模型中被丰富。至关重要的是, 要让系统达到稳定的状态, 在这种状态下, 社区和产生的代谢物保持稳定。本手稿的实验结果证明了接种肠道微生物群群落如何随着时间的推移发展成为一个稳定的群落。一旦达到稳定状态, 该系统可用于分析细菌相互作用和社区功能, 或测试任何添加剂对肠道微生物群的影响, 如食品、食品成分或药物。

上述协议描述了一个5个阶段的体外系统的建立、接种和运行, 以研究结肠的肠道微生物群。为了生成以下数据, 在 dna 提取后, 儿童医院微生物组中心利用高通量测序 (例如 miseq ilumina 平台) 对 v1v2区域进行了 16s rRNA 标记基因 dna 测序费城27。采用 qiime (微生物生态学定量洞察)1.9 28 版对测序数据进行处理, 并在 r 环境下进行统计分析, ...

Watch this Scientific Journal Video about Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota at JoVE.com

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

应用先进的体外培养技术研究人肠道微生物群

在这里, 我们提出了一个在体外培养结肠肠道微生物群的方案,使用一系列生物反应器, 模拟胃肠道的生理条件。

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

该协议的目标是在体外培养肠道微生物群。它将允许研究微生物群动力学，而不必考虑现代成分的贡献。利用这个多阶段体外系统，我们计算微生物群落在发光和多区域的结肠可以同时启动的微生物群落。由于体外系统的复杂性，此方法的视觉演示非常有用。首先用500毫升的已定义介质填充上升冒号，用800毫升的已定义介质填充横向冒号，用600毫升的已定义介质填充下降冒号。将粘液加在结肠区域，其量与反应器的体积成正比，并使用样品管和注射器从每个反应器中去除任何多余的定介质。使用 pH 探头调整每个反应器中的 pH。打开氮气冲洗20分钟。接下来，在用粪便均质样品装入60毫升注射器之前，根据制造商的指南解冻粪便均质。然后，通过样品端口为每个结肠反应器接种相当于每个反应器体积的5%的均质量，用于隔夜培养pH值控制。第二天早上，用酒精清洁每个生物反应器样品口的卢尔锁。当端口干燥后，将 30 毫升注射器（清除所有空气）连接到其中一个样品端口。在从生物反应器中吸入15毫升的培养物超生体之前，将柱塞提取三到五次，以确保容器成分的彻底混合。然后将发光样品转移到无菌的猎鹰管中，放在冰上。对于DNA分析，使用五毫升注射器收集一到三毫升的发光液。以相同的方式收集其他生物反应器的超生形式。为了留出样品材料进行短链脂肪酸分析，将15毫升的发光样品用于进行分离，通过0.2微米孔隙过滤器将超盐过滤，用于零下80摄氏度的储存。为了留出样品材料进行DNA分析，将一毫升发光液离心机，将颗粒储存在零下80摄氏度。对于粘膜样品的收获，从四摄氏度的储存中去除准备好的粘液载体，然后打开氮气冲洗。快速打开反应器盖，用新反应堆更换每个反应堆中 50% 的车厢。更换所有托架后，快速重新密封盖子，将氮气冲洗再保持 20 分钟。然后等分0.25至0.5克粘液载体的阿加到个别两毫升管中，储存零下80摄氏度直到DNA提取。系统每天三次自动循环。这从140毫升的确定介质转移到胃生物反应器，然后一个小时的孵育与pH控制开始。在孵育结束时，胃中的内容与60毫升胰腺汁一起被转移到小肠中。在小肠生物反应器中，pH值自动调整为6.7至6.9，内容物孵育90分钟。在此孵育期间，每个系统的氮冲洗将打开共10分钟。在此孵育结束时，从小肠中的内容被转移到上升结肠中，从上升结肠转移到横向结肠，从横向结肠转移到下降结肠，从下降结肠转移到废物。在这个代表性的主要坐标分析中，社区在第一天和第七天之间发生了贬值变化。然而，从接种后第11天到实验结束，样本占据了一小块区域的主要坐标分析空间，为两个样本样本提供基于操作分类单位、存在到不存在和丰度的样本距离分数。对三种突出的短链脂肪酸的测量表明，丙酸、巴诺酸和醋酸从实验开始一直到接种后第15天波动。第15天之后，每个结肠区域产生的这些酸的含量保持不变，在实验结束之前变化很小。对每个结肠区域的发光和粘膜相的稳定群落的分析表明，在体外系统各个结肠区域发育的群落与粪便的内分组成相似。对体外系统的α多样性的比较揭示了与除上升区域的发光样本以外的所有区域的入体多样性水平。证明体外培养系统能够在组成和多样性方面产生可与粪便的致病社区。要使用此协议产生稳定的肠道微生物群落，必须确保准确准备溶液，并确保在整个实验中保持厌氧条件和pH参数。按照此过程，可以使用下一代DNA测序分析社区成分样本，然后进行生物信息分析，并使用代谢组学分析功能。

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

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循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

在小型组织培养体系培养老鼠心脏瓣膜

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

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眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

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

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

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This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

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用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...