Name: assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface
Uploaded: 2018-07-04T13:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

作者感谢佛兰德研究基金会对玛尔塔卡拉塔余德 (FWO 博士后 fellowship-12N2815N) 的财政支持。艾玛-加西亚萨纳布里亚是一个博士后研究员支持的富兰德创新和企业家精神 (Agentschap 与客厅里 Innovatie 门 Wetenschap créative, 内陆水运)。

1. 菌株和文化条件
注: 综合口腔群落由口腔微生物群中常见的菌株组成3。
<ol><li>从美国类型文化收集 (ATCC) 获得以下菌株: Aggregatibacter actinomycetemcomitans (ATCC 43718),梭杆菌 nucleatum (ATCC 10953),牙龈菌(ATCC33277),普氏菌中间体(ATCC 25611), 变形链球菌 (ATCC 25175),链球菌远缘(ATCC 33478),放线菌对内氏(ATCC 51655), 仙人掌链球菌 (ATCC 49818),放线菌放线菌(ATCC 15987) 和链球菌缓症(ATCC 49456)。</li>
	<li>获得Veillonella parvula从莱布尼茨学院 DSMZ-德国收集的微生物和细胞培养 (DSM 2007),链球菌血统从 BCCM/LMG 细菌收集 (LMG 14657) 和使用链球菌唾液菌株 TOVE-R9, 口腔链球菌 10。</li>
</ol>2. 开发多种群社区代表口腔微生物群
注: 生成合成群落, 模拟口腔细菌对体外肠道上皮的潜在黏附能力。在血液琼脂板上生长细菌补充与5微克/毫升血红, 1 微克/毫升甲萘醌, 5% 无菌 defibrinated 马血, 如埃尔南德斯-加西亚萨纳布里亚等。(2017)11. 简言之:
<ol><li>将100毫克的血红溶解在2毫升的1米氢氧化钠中, 加入100毫升的蒸馏蒸压水。储存在一个黑暗的容器里。在添加到培养基前, 通过0.22 µm 过滤器进行消毒。</li>
	<li>在20毫升96% 乙醇中溶解100毫克的甲萘醌。在添加到培养基前, 通过0.22 µm 过滤器进行消毒。</li>
	<li>根据制造商的说明和高压釜, 准备1升的血液琼脂培养基 (见材料表)。在添加马血和补充剂之前, 让冷却下来。混合并倒入盘子。</li>
	<li>准备改良的脑心输液 (BHI) 汤, 如前所报告的12。在热处理后加入5微克/毫升的血红和1微克/毫升甲萘醌。</li>
	<li>在亨盖特管中分配9毫升的培养基, 并与橡胶塞子和铝盖紧密结合。用 N2/CO2 (90%/10%) 冲洗管子。</li>
	<li>用注射器检索1毫升厌氧培养基, 并将其放在1.5 毫升的离心管中。选取每个细菌种类的单一菌落, 在培养基中并用重悬, 然后转移回厌氧改性 BHI。孵化48小时在37摄氏度, 除了S. 唾液, 必须孵化24小时。</li>
	<li>
如果需要, 在组装合成群落之前确认文化的纯度。简言之:
	<ol>
<li>从液体培养中提取 DNA, 如加西亚萨纳布里亚等. (2010)13和放大 16S rRNA 基因使用引物 27F (AGAGTTTGATYMTGGCTCAG) 和 1492R (TACGGYTACCTTGTTACGACT) 和以下节目: 5 分钟在95°c, 30 个周期95°c 为1分钟, 55 °c 为1分钟和72°c 为1.5 分钟, 然后最后的延长步5分钟, 在72摄氏度。</li>
		<li>按照制造商的说明, 用商业套件 (见材料表) 净化 PCR 产品。</li>
		<li>在 10 ul 总容积中, 执行纯度为0.5 的染料 (见材料表)、3.2 pmol M13f 底漆 (CGCCAGGGTTTTCCCAGTCACGAC)、2.0 ul 5x 序缓冲器和 20 ng 模板的纯化产品的排序反应, 使用商业测序系统用套件 (参见材料表)。 注意: 我们在商业上执行了这个程序。</li>
		<li>在所有序列 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) 上执行一次爆破搜索, 以确定最接近已知的分类, 并使用 RDP 分类器在线工具 (http://rdp.cme.msu.edu/) 和新浪与原始应变序列进行比较。对齐服务 (https://www.arb-silva.de/aligner/)。</li>
	</ol>
</li>
	<li>用流式细胞仪和 SYBR 绿/碘碘染色等方法在孵化末期测量细胞数, 加西亚萨纳布里亚等。(2017)11. 利用改良的 BHI 将培养物稀释为 105细胞毫升-1。</li>
	<li>加入1毫升的缓症到75毫升的厌氧 BHI 和孵化直到固定相 (6 小时)。以下, 收集0.75 毫升的每稀释应变和混合在厌氧条件下。</li>
	<li>一旦所有的文化混合, 采取1毫升的缩影, 并添加到75毫升的 BHI。孵化合成社区在 10% CO2, 10% H2, 80% N2为 48 H 在37°c 和 200 rpm (参见材料表)。</li>
	<li>测量细胞密度如2.8。在向细胞模型展示社区之前。使用斯洛姆卡等表1中的引物和退火温度, 可对群落组成进行定量实时 PCR 评价。(2017)10. 此外, 使用 16S rRNA 基因扩增子测序来提供有关最初成员13、14的信息。对于两种身份确认协议, 使用 cDNA 作为模板, 表明细菌积极转录蛋白, 并使用 DNA 作为模板, 以显示存在/没有的菌株。</li>
</ol>3. 一般细胞培养做法
注: 对于细胞培养的一般方面, 作者指的是硕士和史黛西 (2007)15。获得从欧洲收集的经鉴定的细胞培养 (Caco-2 ECACC 86010202 和 HT29-MTX-E12 ECACC 12040401, 英国公共卫生) 使用的细胞线。除非另有规定, 细胞培养试剂可以购买 (见材料表)。
<ol><li>
Caco-2 和 HT29-MTX 细胞系的维护与传代 注: Caco-2 和 HT29-MTX 细胞通常生长在25厘米2的组织培养烧瓶中, 含有补充的 DMEM 培养基 (表 1)。当60–70% 汇合到达时, 细胞 trypsinized。<ol>
<li>从烧瓶中取出细胞培养基。清洗两次与5毫升 PBS 没有 Ca ++/毫克+ +。</li>
		<li>加入2毫升的0.5 毫克/升溶液的胰蛋白酶和0.22 克/l-乙烯二胺四乙酸四酸 (EDTA)。通过轻轻地移动烧瓶来分发胰蛋白酶溶液。去掉1.5 毫升的胰蛋白酶溶液, 在37摄氏度孵化出10分钟的烧瓶。</li>
		<li>在显微镜下检查细胞是否与瓶子表面分离。如果需要, 孵育额外的5分钟。</li>
		<li>添加5毫升的补充细胞培养培养基, 以使胰蛋白酶钝化。吸管向上和向下, 以获得一个均匀的单细胞悬浮。</li>
		<li>立即, 采取50µL 整除细胞悬浮和混合与无菌0.4% 台盼蓝溶液在 1:1 (v/v) 比例为 Caco-2 细胞或1:5 伏/五为 HT29-MTX。混合吹打和负载到一个计数室 (见材料表)。</li>
		<li>在显微镜下计数可行的细胞 (白色明亮的细胞) (见制造商的说明)。</li>
		<li>种子在一个新的烧瓶密度为 4 x 105细胞/cm2和 1 x 104细胞/厘米2, 分别为 Caco-2 和 HT29-MTX。 注: (1) Caco-2 细胞分化并形成紧密连接, 一旦到达融合。在 trypsinization 之前避免过度融合是非常重要的。trypsinization 和/或细胞团簇后, 烧瓶细胞过度生长会导致细胞脱离。(2) HT29-MTX 细胞为粘液生成细胞, 代谢活性高16。这些特征可能导致细胞培养基的快速酸化, 特别是当细胞处于高密度时。HEPES 缓冲可以添加到细胞培养基 (表 1) 中, 以保持 pH 值在7和7.2 之间。如果 pH 值下降, 培养基可以在融合小于50% 时进行交换。但是, 如果融合 > 50%, 细胞培养必须分裂。</li>
	</ol>
</li>
</ol>4. 模拟肠道宿主-微生物界面的 Multicompartment 细胞模型的组装
注: 完成双腔井共培养 (直径24毫米, 孔径0.4 微米; 见材料表)。
<ol><li>添加1.5 毫升完整的细胞培养基到顶端的隔间和2毫升到基部。预孵化20–30分钟, 以获得均匀分布的细胞悬浮时, 播种。</li>
	<li>胰蛋白酶处理 Caco-2 和 HT29-MTX 的细胞培养, 如第3节所述。trypsinization 程序必须准确地遵循, 以实现两个细胞系的均匀分布, 在单个细胞悬浮, 没有团簇。</li>
	<li>按3.1.8 中所述计算可行细胞数。</li>
	<li>将细胞密度调整为 6.5 x 104细胞/cm2 , Caco-2/HT29-MTX 细胞的90/10 比例。</li>
	<li>融汇细胞悬浮液, 并将相应体积的 Caco-2 和 HT29-MTX 细胞添加到预填充的无菌容器中, 并辅以细胞培养培养基。将预孵化的介质从腔室的顶端侧取出。</li>
	<li>用吹打轻轻地混合细胞悬浮液, 并将1.5 毫升转移到腔体插入物的顶端隔间。播种过程需要恒定的均匀化细胞悬浮, 因为细胞倾向于沉淀物。根据用户的经验和工作速度, 在配药1–3井后必须混合细胞悬浮液。</li>
	<li>移动板块轻轻向后和向前, 然后从右向左向右 (5–10倍), 以获得均匀扩散的细胞在井中。转移到孵化器和重复的板块移动。</li>
	<li>保持 Caco-2/HT29-MTX 细胞的共培养15天, 刷新顶端和基底隔间每2天与补充 DMEM。在此之后, 细胞培养培养基可以改变为补充 DMEM 没有抗生素/防霉溶液。</li>
	<li>保持系统, 直到20–21天播种后, 每2天刷新培养基。</li>
	<li>
使用电阻系统 (见材料表), 按照制造商的指示, 测量上皮屏障的完整性。
	<ol>
<li>在开始测量之前, 确保志愿者设备全部充电 (> 24 小时)。</li>
		<li>从电源上拔下志愿者设备并盖上塑料蒸压袋。在袋子里打一个洞, 介绍电极并插入仪表上的输入端口。喷洒70% 乙醇/水 (v/v), 然后将志愿者设备引入流柜。</li>
		<li>要对电极进行消毒, 请在70% 乙醇/水 (v/v) 溶液中浸泡电极提示15分钟, 使空气干燥十五年代. 在预热细胞培养基中冲洗电极。</li>
		<li>将细胞从孵化器中取出, 并允许细胞在流动柜内进入室温。</li>
		<li>请确保仪表与充电器断开。将模式开关设置为欧姆, 然后打开电源开关。</li>
		<li>通过将电极浸泡在插入物中, 用较长的尖端从井中浸出, 测量细胞电阻。将电极保持在90°角到板插入。</li>
	</ol>
</li>
</ol>5.体外胃肠道转运条件下的细菌存活
注: 制备模拟消化液后, Minekus等协议。(2014)17、下面描述的修改。用超纯水制备所有的稀释。过滤-通过0.22 µm 过滤器消毒所有解决方案, 并在无菌条件下执行消化过程。
<ol><li>
口腔消化:
	<ol>
<li>在50毫升不育管中放置3毫升细菌群落, 与3毫升模拟唾液液混合, 含 (在毫摩尔 L-1): 氯化钾, 15.1;2PO4, 3.7;NaHCO3, 6.8;氯化镁2(H2O)6, 0.5, (NH4) CO3, 0.06;HCl, 1.1;CaCl2(H2O)2, 0.75。添加75毫升的α淀粉酶从人类唾液类型 IX A 和2克/升黏蛋白从猪胃 ii. 型。</li>
		<li>孵育混合物2分钟, 在37摄氏度和 100 rpm。收集2毫升的 DNA 提取和流式细胞仪定量 (S1) 样本。</li>
	</ol>
</li>
	<li>
胃消化:
	<ol>
<li>添加4毫升模拟胃液 (SGF), 含 (在毫摩尔 L-1): 氯化钾, 6.9;2PO4, 0.9;NaHCO3, 25;氯化钠, 47.2;氯化镁2(H2O)6, 0.1, (NH4) CO3, 0.5;HCl, 15.6;CaCl2(H2O)2, 0.075。此外, SGF 中含有2克/升的猪胃 ii. 型粘蛋白。</li>
		<li>如果需要, 测量 ph 值并调整为 ph 值3和1米 HCl。孵育在37°c 和100转每分钟。收集2毫升样品 (S2)。</li>
	</ol>
</li>
	<li>
小肠消化:
	<ol>
<li>添加4毫升模拟肠道液 (SIF), 含 (在毫摩尔 L-1): 氯化钾, 6.8;2PO4, 0.8;NaHCO3, 85;氯化钠, 38.4;氯化镁2(H2O)6, 0.33;HCl, 8.4;CaCl2(H2O)2, 0.3;消化管。</li>
		<li>如果需要, 调整到 pH 值7与1米氢氧化钠。孵育在37°c 和100转每分钟。收集2毫升样品 (S3)。</li>
	</ol>
</li>
</ol>6.体外胃肠道转运条件下的细菌定植能力
<ol><li>离心2毫升小肠消化, 10 分钟在 2650 x g。</li>
	<li>除去上清, 并将细菌颗粒与5毫升补充 DMEM, 无抗生素和防霉。</li>
	<li>使用流式细胞仪 (参见材料表) 染色并量化完整/损坏的单元格, 如加西亚萨纳布里亚等所述。(2017)11。</li>
	<li>在没有抗生素和防霉的情况下, 通过稀释补充 DMEM, 调节细胞密度为 105活细胞/毫升。</li>
	<li>去除 transwell 系统的顶端介质, 加入1.5 毫升的细菌悬浮液。共同养殖系统为 2 h 在一般细胞培养情况下 (37 °c, 95% 湿气, 5% CO2)。 注: 此时间可扩展到 48 h, 具体取决于所需的实验协议。共同培养可以维持在不同的孵化器, 而不是常规的细胞维护, 以避免污染。</li>
	<li>在孵化后测量志愿者值, 以评估4.11 中所描述的上皮屏障的完整性。</li>
	<li>完成潜伏期后, 取出顶端培养基, 收集0.5 毫升作进一步分析 (S4)。在制造商的指导下, 亚组的培养基可以用来评估乳酸脱氢酶 (LDH) 测定的细胞活力 (见材料表)。</li>
	<li>在 HBSS (NAC 缓冲器) 中加入0.5 毫升10毫米 n-乙酰半胱氨酸, 溶解 HT29-MTX 产生的粘液, 并恢复附着的细菌。孵育板材在37°c 为 1 h, 在鼓动下 (135 rpm, 看见材料表)。</li>
	<li>恢复离心管 (S5) 中的 NAC-HBSS, 并用1毫升 DPBS 清洗两次细胞。</li>
	<li>添加0.5 毫升0.5% 海卫 X-100 (瓦特/v) 在单层膜上, 通过吹打扰乱细胞, 恢复液体和漩涡1分钟. 速度对于避免用洗涤剂破坏细菌细胞是至关重要的。</li>
	<li>将细胞离心5分钟, 在 2650 x g处分离出上清 (S6) 和颗粒 (S7)。</li>
</ol>7. 样品分析
注: 分析收集的样品 (S1-S7), 以评估细菌的生存能力和黏附能力的肠道细胞, 经过模拟胃肠消化。
<ol><li>细菌生存能力: 通过样品 S1-S5 两次通过0.45 µm 和量化细菌细胞使用活/死细胞染色和流式细胞术, 如步骤 2.811所示。</li>
	<li>
粘附电位: 为 S1-S6 和条纹10µL 在血琼脂和改良 BHI 板上准备连续稀释 (-1 到-8)。在厌氧条件下孵育37摄氏度, 24–48的 S7。
	<ol>
<li>粘附细菌的分类鉴定:<ol>
<li>选择一个文化循环的个体殖民地, 并并用重悬在10µL 的核酸酶无水。收集4µL 的悬浮和使用它作为一个模板, PCR 扩增的 16S rRNA 基因使用引物和条件在2.7.1 描述。</li>
			<li>按照2.7.3 中的协议执行排序, 并使用2.7.4 中包含的过程验证序列。</li>
		</ol>
</li>
	</ol>
</li>
	<li>做进一步的下游分析, 如总 DNA 提取, qPCR 量化和/或 16S rRNA 扩增子测序, RNA 提取, 和 transcriptomic 分析的期望。 注: 在取样后立即进行流式细胞仪定量。作为对膜透细胞的一种控制, 使用了70摄氏度的样品进行3分钟的接触。利用流式细胞仪测量无细菌细胞培养模型中的消化液或样品, 对背景噪声进行了评价。每样样品均用三份计量。</li>
</ol>

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作者没有什么可透露的

口服微生物菌群是人类健康的一个关键因素, 最近有几个作者20、21发表了报告。先前的研究表明, 摄取含有大量细菌的唾液会影响小肠的微生物生态系统, 这是免疫启动的主要部位之一。静态上消化道消化模型与由肠道上皮和粘液分泌细胞代表的宿主界面的结合, 有助于解开微生物成分对宿主的影响。
体外模型是研究的基本工具, 允许进行机械研究和获得背景知识, 旨在减少, 使更有效率, 更好的目标在体内研究。因此, 在建立合成微生物群落之前, 确保无菌文化的纯度、活力和最佳生长至关重要。我们建议在接触细胞培养之前评估细菌群落的生存能力和组成, 以避免分析中的偏见。大量受损细菌可能会阻碍粘连, 进而影响细胞模型的完整性。此外, 使用可信的细胞线来源将减少细胞线误认, 污染和不良的注释, 提高实验重现性22。
包括粘液生成细胞使相似的小肠, 因为粘液和粘液提供第一防御线的胃肠道23。此外, 粘膜层适用于细菌的定植, 允许表征的双侧转运细菌代谢物。此外, 模拟胃肠道条件增加了乳酸杆菌 paracasei菌株对 Caco-2 和粘蛋白24的黏附能力, 支持在我们的模型中纳入消化过程的相关性。
该模型的进一步改进可以引入免疫宿主组件, 如前所述25。然而, 目前的上皮和免疫细胞共培养系统尚未用于宿主-微生物相互作用的应用 (例如,对食品生物活性化合物或益生菌的评价), 这可能表明, 这些系统可能缺乏重现性25。
以前的研究与 Caco-2, HT29-MTX 或联合文化评估了益生菌或致病菌株的黏附力对哺乳动物细胞26,27。使用单一污渍或少量微生物的关联可能对宿主-微生物相互作用提供有限的概述, 它并不代表人类胃肠道的复杂性。虽然使用天然微生物群落可以更有代表性的在体内的情况下, 他们是难以描述和研究。相比之下, 我们的方法提供了评估多宿主-微生物相互作用的机会, 使用一个复杂和有代表性的缩影。使用定义的合成微生物群落可以生成一个降低复杂性28的系统。社区的变化可能是个体实验背景下微环境条件的结果。因此, 该模型的微生物成分可以很容易地放大或下降, 以解构环境的影响作为一种选择性的力量。最后, 采样辅助功能保证了人类细胞和相关微生物群落功能活动的进一步描述。
近年来, 在 organoid 技术的基础上, 建立了新的体外模型。Organoids 是3D 细胞培养, 它融合了代表的主要组织的一些主要特征。肠道 organoids 是研究成体干细胞和组织的近生理系统, 但这种模型也有一定的局限性。肠道 organoids 对感染或药物的炎症反应的使用有限, 因为它们缺乏免疫细胞。此外, organoids 的可行性, 大小和形状, 阻碍表型筛选和渲染标准化复杂的异构。尽管在健康组织体外建模中, 永生化细胞系受到限制, 但使用具有良好特征和稳定性的细胞系也具有重复性、重现度和低成本等优点。这些好处允许同时筛选几个条件, 但对数据的平移使用是有争议的。原代细胞在体内生理上可能更具代表性;但是, 它们具有很高的个体间变异性, 没有充分的特征, 是异构的, 而且时间跨度有限。这些因素是一个缺点, 包括多种条件或复制在一个化验。
结合细胞系与复杂的微生物群落可能是一个挑战, 我们的重点是开发一个可重复和可复制的模型, 具有较高的灵活性和适用性, 在实验室与基本细胞设备, 直到更具代表性模型变得可利用和好描绘。我们用这些 multicompartment 模型评估了不同细菌群落的功能, 例如, 评估上消化道的益生菌行为和表征 xenobiotic 对肠道界面的影响。因此, 我们建议这个模型作为一个基础, 以进一步化验与广泛的各种益生菌, 药物, 或食品化合物, 在一个相关的和定义的体外生态系统。

人与细菌同居, 是存在与人细胞1的同一个数字。因此, 对人类微生物群的全面了解是至关重要的。口腔是一个独特的环境, 因为它被分成几个较小的栖息地, 从而包含了大量的细菌和生物膜在这些不同的地方。作为一个开放的生态系统, 一些物种在口中可能是短暂的访客。然而, 某些微生物在出生后不久就会殖民, 形成组织的生物膜2。这些在牙龈缝隙、龈下缝隙、舌头、粘膜表面和牙科修复体和填充物3中的牙齿表面被发现。细菌也可以作为絮凝体和浮游细胞存在于牙管腔内, 或者与坏死的牙髓组织混合或悬浮在液相中。
宿主细胞与居民微生物群之间有积极、连续的交叉谈话4。细菌在物种内部和之间进行交流, 只有很小一部分自然殖民者能够坚持组织, 而其他细菌则依附于这些主要殖民者。例如, 微生物之间的细胞结合是将次级殖民者纳入口腔生物膜的关键, 并建立相互作用的微生物细胞的复杂网络4。唾液样品中大约70% 的细菌聚集物由牙龈sp、普氏菌链球菌、 Veillonella sp 和不明Bacteroidetes组成。nucleatum是龈下生物膜中的中间体殖民者, 与晚期殖民者菌、denticola和Tannerella 连翘有牵连, 这与牙周炎5有关。此外,缓症链球菌还占据粘膜和牙科栖息地, 而血统和仙人掌则更倾向于将牙齿殖民3。因此, S. 血统是存在于下门牙和犬齿, 而放线菌对内氏已发现在上部 anteriors6。
此外, 土著微生物群在维护人类健康方面起到了2的作用。居民微生物参与免疫教育和防止病原体扩张。这种殖民抵抗的发生是因为当地的细菌可以更好地适应于附着在表面上, 并且更有效地代谢可利用的养分促进生长。虽然益生菌菌株在胃肠道中存活并保持活跃, 但仍未充分描述从胃肠道上部位置吞下的本土细菌的持久性。因此, 我们接受了一个人工群落, 代表口腔生态系统, 模拟胃肠道转运条件。用类似肠道上皮的 multicompartment 模型评估细菌细胞的生存能力。目前肠道模拟器提供了适当的重现性分析的腔内微生物群落7。然而, 细菌黏附力和寄主微生物相互作用是分开解决的, 因为结合细胞系与微生物群落是挑战8。相反, 我们提出了一个框架, 提供了潜在的机械解释的成功的殖民事件报告的肠道接口。事实上, 这个模型可以与静态肠道模型联合使用, 以评估微生物群落对宿主表面信号的影响。

<table><tbody><tr><td>&lt;strong&gt;STRAINS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Aggregatibacter actinomycetemcomitans &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 43718</td><td></td></tr><tr><td>&lt;em&gt;Fusobacterium nucleatum &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 10953</td><td></td></tr><tr><td>&lt;em&gt;Porphyromonas gingivalis &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 33277</td><td></td></tr><tr><td>&lt;em&gt;Prevotella intermedia &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 25611</td><td></td></tr><tr><td>&lt;em&gt;Streptococcus mutans &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 25175</td><td></td></tr><tr><td>&lt;em&gt;Streptococcus sobrinus &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 33478</td><td></td></tr><tr><td>&lt;em&gt;Actinomyces viscosus &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 15987</td><td></td></tr><tr><td>&lt;em&gt;Streptococcus salivarius&amp;nbsp; TOVE-R&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Streptococcus mitis &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 49456</td><td></td></tr><tr><td>&lt;em&gt;Streptococcus sanguinis &lt;/em&gt;</td><td>BCCM/LMG Bacteria Collection</td><td>LMG 14657</td><td></td></tr><tr><td>&lt;em&gt;Veillonella parvula &lt;/em&gt;</td><td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures</td><td>DSM 2007</td><td></td></tr><tr><td>&lt;em&gt;Streptococcus gordonii &lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 49818</td><td></td></tr><tr><td>&lt;strong&gt;CELL LINES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Caco-2 cells</td><td>European Collection of Authenticated Cell Cultures</td><td>86010202</td><td></td></tr><tr><td>HT29-MTX cells</td><td>European Collection of Authenticated Cell Cultures</td><td>12040401</td><td></td></tr><tr><td>&lt;strong&gt;REAGENTS AND CONSUMABLES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Brain Heart Infusion (BHI) broth</td><td>Oxoid</td><td>CM1135</td><td></td></tr><tr><td>Blood Agar 2</td><td>Oxoid</td><td>CM0055</td><td>Blood Agar medium</td></tr><tr><td>Menadione</td><td>Sigma</td><td>M9429</td><td></td></tr><tr><td>Hemin</td><td>Sigma</td><td>H9039</td><td></td></tr><tr><td>5% sterile defibrinated horse blood</td><td>E&amp;amp;O Laboratories Ltd,</td><td>P030</td><td></td></tr><tr><td>InnuPREP PCRpure Kit</td><td>Analytik Jena</td><td>845-KS-5010250</td><td>PCR purification kit</td></tr><tr><td>Big Dye</td><td>Applied Biosystems</td><td>4337454</td><td>Dye for sequencing</td></tr><tr><td>ABI Prism BigDye Terminator v3.1 cycle sequencing kit</td><td>Applied Biosystems</td><td>4337456</td><td></td></tr><tr><td>SYBR&amp;nbsp;Green I</td><td>Invitrogen</td><td>S7585</td><td></td></tr><tr><td>Propidium Iodide</td><td>Invitrogen</td><td>P1304MP</td><td></td></tr><tr><td>T25 culture flasks uncoated, cell-culture treated, vented, sterile</td><td>VWR</td><td>734-2311</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T3924-100ML</td><td></td></tr><tr><td>Trypan Blue solution&lt;br/&gt; 0.4%, liquid, sterile-filtered</td><td>Sigma-Aldrich</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190250</td><td></td></tr><tr><td>DMEM cell culture media, with GlutaMAX and Pyruvate</td><td>Life technologies</td><td>31966-047</td><td></td></tr><tr><td>Corning Transwell polyester membrane cell culture inserts</td><td>Sigma-Aldrich</td><td>CLS3450-24EA</td><td></td></tr><tr><td>Mucin from porcine stomach Type II&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M2378</td><td></td></tr><tr><td>Inactivated fetal bovine serum</td><td>Greiner Bio One</td><td>758093</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Triton X 100 for molecular biology</td><td>Sigma-Aldrich</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>DPBS without calcium, magnesium</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Pierce LDH Cytotoxicity Assay Kit</td><td>Thermo Fisher Scientific</td><td>88953</td><td></td></tr><tr><td>Corning HTS Transwell-24 well, pore size 0.4 &amp;micro;m</td><td>Corning Costar Corp</td><td>3450</td><td></td></tr><tr><td>Nuclease-free water</td><td>Serva Electrophoresis</td><td>28539010</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Neubauer counting chamber improved</td><td>Carl Roth</td><td>T729.1</td><td></td></tr><tr><td>BD Accuri C6 Flow cytometer</td><td>BD Biosciences</td><td>653118</td><td></td></tr><tr><td>PowerLyzer 24 Homogenizer</td><td>MoBio</td><td>13155</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>BioRad</td><td>186-1096</td><td></td></tr><tr><td>Flush system</td><td>Custom made</td><td>-</td><td></td></tr><tr><td>InnOva 4080 Incubator Shaker</td><td>New Brunswick Scientific</td><td>8261-30-1007</td><td>Shaker for 2.10</td></tr><tr><td>Memmert CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Memmert GmbH &amp;amp; Co.</td><td>ICO150med</td><td></td></tr><tr><td>Millicell ERS (Electrical Resistance System)</td><td>EMD Millipore, Merck KGaA</td><td>MERS00002</td><td></td></tr><tr><td>Millipore Milli-Q academic, ultra pure water system</td><td>Millipore, Merck KGaA</td><td>-</td><td></td></tr><tr><td>Shaker (ROCKER 3D basic)</td><td>IKA</td><td>4000000</td><td>Shaker for 6.10</td></tr></tbody></table>

assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface

宿主和微生物群之间的相互作用已被长期承认和广泛描述。口腔与胃肠道的其他部分相似, 因为居民的微生物群发生, 防止外源细菌的定植。事实上, 在口腔中发现了600多种细菌, 单个个体在任何时候都可能携带100左右。口腔细菌具有在口腔生态系统中坚持各种利基的能力, 从而在居民微生物群落中得到整合, 有利于生长和生存。然而, 在吞咽过程中细菌进入肠道的流动已经被提出来扰乱肠道微生物群的平衡。事实上, 口服菌转移了回肠菌群中的细菌组成。我们利用合成社区作为自然口腔生态系统的简化表示, 以阐明在模拟胃肠道条件下口腔细菌的存活和生存能力。选择十四种, 经体外唾液、胃和肠道消化过程, 并提出一个 multicompartment 细胞模型, 包含 Caco-2 和 HT29-MTX 细胞模拟肠道黏膜上皮。该模型有助于解开吞噬细菌对肝肠循环细胞的影响。使用合成群落可以进行可控性和重现。因此, 这种方法可以适应评估病原体的生存能力和随后的炎症相关变化, 益生菌混合物的殖民化容量, 最终, 潜在的细菌对 presystemic 循环的影响。

该协议导致了一个模型的生成, 适合于阐明口腔细菌在模拟胃肠道转运条件下的生存和存活。从单个菌株的完整细胞计数约 108细胞 mL-1之前, 合成社区的创建, 而多种群的缩影包含90% 的可行细胞在社区的建立 (图 1A和1B)。根据活/死定量, 细菌的生存能力减少后, 每消化步骤 (图 2)。这可能是由于胃酸 pH 值在胃通道和胆汁盐包含在小肠消化液, 如发生在生理条件。在通过体外和体内消化道消化过程中的生存能力可能取决于环境的变化 (例如,喂食与禁食条件), 甚至是细菌保护过程 (孢子形成或肠道涂层)。
流量细胞计数必须与适当的控制, 如热杀死细菌或背景样品, 以执行准确可行的细胞量化18。胃和小肠消化的严酷条件可以将细菌转变为可行但非可培养的细胞, 而不是生长或分裂的活细菌。因此, 板块计数不同于用流式细胞仪获得的活细胞计数。例如, 在图 3中, 在流式细胞术图中, 从粘膜界面中恢复的存活细胞呈蓝色。然而, 当这些粘液样品被镀 (结果未显示) 时, 没有观察到生长。
细菌表现出更高的生存率的分数是细胞碎片 (S7), 如板块计数所揭示的 (图 4)。菌落数量可能是可变的, 但在稀释的样品中发现有代谢活性细菌的存在。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57699/57699fig1.jpg"> 图 1:在体外消化消化初期, 多种群微生物群落组成的细胞生存能力。 (A)活/死染色和流式细胞术 (平均标准偏差, n = 3) 量化的可行细胞计数 (测井单元)。(B)在共培养48小时后, 多种群微生物群落的代表性流式细胞仪图。绿色、红色和黑色的斑点分别代表可行的、受损的和背景的或非分类的细菌。<a href="https://www.jove.com/files/ftp_upload/57699/57699fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57699/57699fig2.jpg"> 图 2: 模拟胃肠消化后细菌的细胞生存能力.在口服、胃和小肠消化步骤 (平均标准偏差, n = 3) 后, 条形表示存活细胞 (对数单位) 的数量。<a href="https://www.jove.com/files/ftp_upload/57699/57699fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57699/57699fig3.jpg"> 图 3: 粘膜间界面存活细菌细胞的代表性图.红点代表背景和蓝色点的可行细胞。基于道具等方法建立了流式细胞术的条件。(2016)19. 粘膜样品稀释 1:100 (v/v), 染色 Sybr 绿色/碘碘化物15分钟, 100 µL 的样品测量在 FL1: 533/30 nm, FL2: 585/40 nm, FL3: > 670 nm 长通行证, 和 FL4: 675/25 nm。<a href="https://www.jove.com/files/ftp_upload/57699/57699fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57699/57699fig4.jpg"> 图 4: 在改性 BHI 板上生长的细胞碎片的菌落形成单元 (CFU).三生物复制用于细菌粘附试验, 并进行了三项技术复制 CFU 量化。图中显示了两个包含-1 到-6 稀释的细胞碎片 (S7) 的复制板。<a href="https://www.jove.com/files/ftp_upload/57699/57699fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>细胞培养</td>
			<td>粘附/悬挂</td>
			<td>培养基培养</td>
			<td>一般补充剂</td>
			<td>特殊补充剂</td>
			<td>加倍时间</td>
			<td>孵化条件</td>
		</tr>
<tr>
<td>Caco-2 (ECACC 86010202)</td>
			<td>粘 附</td>
			<td rowspan="2">Dulbecco 改性鹰培养基 (DMEM), 4.5 克/升葡萄糖</td>
			<td rowspan="2">胎牛血清灭活 (10% 伏/五) 青霉素 (100 U/毫升) * 链霉素 (0.1 毫克/毫升) * 两性霉素 (0.0025 毫克/毫升) *</td>
			<td>Glutamax (4 毫米) 非必需氨基酸 (1% 伏/v) 丙酮酸 (1 毫米)</td>
			<td>65–72 h</td>
			<td rowspan="2">95% 湿气和 10% CO2 CO2孵化器 (参见材料表)</td>
		</tr>
<tr>
<td>HT29-MTX (ECACC 12040401)</td>
			<td>粘 附</td>
			<td>HEPES (1 毫米)</td>
			<td>20–24 h</td>
		</tr>
<tr>
<td colspan="7">* 在开始化验前2天, 将抗生素和抗真菌药物从细胞培养培养基中取出。</td>
		</tr>
</tbody></table>表 1: 细胞培养维护的细胞系和培养基组成的描述。

Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

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

采用综合口服群落、体外胃肠消化和小肠上皮模型相结合的新方法对肠道宿主-微生物相互作用进行评价。我们提出了一种方法, 可以适应评估细胞入侵的病原体和多物种生物膜, 甚至测试益生菌制剂的生存性。

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 ...

assessing-viability-synthetic-bacterial-consortium-on-vitro-gut-host

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

11.5K 次观看.  Ghent University. 采用综合口服群落、体外胃肠消化和小肠上皮模型相结合的新方法对肠道宿主-微生物相互作用进行评价。我们提出了一种方法, 可以适应评估细胞入侵的病原体和多物种生物膜, 甚至测试益生菌制剂的生存性。

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

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

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

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 ...

科研

生物工程

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

免疫与感染

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

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 complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

应用先进的体外培养技术研究人肠道微生物群

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 ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

评估介入疗法对人体粪便微生物群影响的体内批处理培养模型

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

分析宿主-微生物群相互作用的肠道肠道器官培养系统

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

生物学

从根际土壤中开发协同多物种生物膜

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

作者聚焦:开发合成微生物群落以生产第二代生物肥料

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

一种从根际土壤构建多物种生物膜群落的方法

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...