Name: human placental and decidual organ cultures to study infections at the maternal-fetal interface
Uploaded: 2016-07-21T15:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface at JoVE.com

We are grateful to Cristina Faralla and David Lowe for helpful discussions. We acknowledge Mark Weinstein and San Francisco General Hospital Pathology Department for expert advice. This work was supported by National Institute of Health grants R01AI084928 and Burroughs Wellcome Fund 41259 to A.I.B; G.A.R. was supported by F32AI108195, Society for Pediatric Pathology Young Investigator Research grant, and University of California Partnerships for Faculty Diversity President's Post-doctoral Fellowship.

实验是根据赫尔辛基宣言中所表达的原则进行。这项研究是经旧金山（CHR＃11-05530）机构审查委员会在加州大学。提供的所有患者写给样品和后续分析收集的知情同意书。组织收集作为去标识标本。 注:研究人员，必须获得其机构人类受试者审查委员会的批准。标本根据妊娠史或胎盘异常排除会根据任何具体研究的需要而有所不同。 注意:适当的安全条件（生物安全2级）应被用于在该协议12的所有步骤。 1.准备，在此之前收集日<ol><li>高压灭菌过滤/收集镊子和微型剪刀解剖/镊子。 </li><li>准备洗涤液的货量充足（500毫升每个试样）（重FER 表1配方）。 </li><li>准备充足的量（每片100毫升）中0.22微米的无菌过滤收集媒体（ 见表1配方）。 </li><li>等分细胞外基质（ECM，参照试剂的表的信息），用于长期存储。 </li><li>商店的ECM以固体形式在-20℃下。为了获得最佳性能，避免重复冻融。解冻瓶在4℃的粘稠溶液，并在冷室用冷却枪头制备300μl的等分试样。商店等分于-20℃。 </li></ol> 2.新鲜组织收集注意:当与新鲜的人体组织时，研究人员必须遵循预防血源性病原体的传播普遍预防（OSHA），且必须已收到适当的机构培训。适当的个人防护装备，包括手套，保护眼睛，和实验室大衣是必要的。 <ol><li>运输蒸压型过滤（每​​样本之一），高压灭菌，镊子，洗涤缓冲液（大玻璃瓶），收集培养基（每个试样25毫升在50ml锥形管），喷雾瓶，10％的漂白剂，70％的乙醇，组织收集托盘，Sharpie笔和冰桶/冷却器医院/诊所。 注:样品在位于接近工作套房一间研究指定的空间接收。的无菌组织培养罩不是必须的，但应尽快发生收集和研究者应适当消毒的表面，如下所述。 </li><li>洗的地方大玻璃瓶缓冲相邻的水槽，并用10％漂白剂和70％的乙醇喷雾轴颈。在光源（光垫或光盒）代替玻璃组织收集盘，以及消毒，用10％漂白剂和70％的乙醇。风干。 </li><li>具有操作套件标本准备室医生随身携带的标本。在宿，倒顶上无菌手持式过滤器样品，冲洗组织数次被H缓冲液，并在缓冲传输整个标本消毒玻璃盘顶上的光源。 注意:由临床医生组织的评估是最高优先级，从而后临床医生已经口头表明他/她完成了评估以下步骤仅执行。 </li><li>使用无菌镊子选择用于收集胎盘绒毛和蜕膜（ 图2A和3A），并且转移到分离50ml锥形管中。标签与胎龄管。 注意:首选胎龄是小于9周，小于16周的胎盘分别和蜕膜。在该机构中，只有每个试样的一小部分采购用于研究目的，因为足够的组织必须保持为临床病理诊断。 </li><li>冰的研究实验室的存储和运输标本。 </li><li>收集多个样品，消毒玻璃盘上方，用10％漂白剂和70％的乙醇的山口之间所描述每个试样的ction。 </li></ol> 3.器官培养板的制备注意:应该在组织培养罩使用无菌技术来执行下面的步骤。它是理想的解剖显微镜将设在一个无菌区域。然而，如迅速进行显微解剖，并且仪器在70％乙醇经常浸入这是不是只要绝对必要的。 <ol><li>解冻ECM样品瓶上冰。稀释ECM 1:1用冰冷的作品集媒体，吹打混匀。吸管慢慢地避免引入气泡。 6孔组织培养板的各孔中，需要100微升该悬浮液和将容纳3-4器官培养。 </li><li>地方的transwell插入物（孔径0.4微米）到6孔组织培养板的每个孔中。 </li><li>涂层的每个Transwell小与ECM /媒体悬浮液的薄层（100微升）插入。 </li><li>广场上冰板立即进行establishmen器官培养的吨。可替代地，准备前组织收集板，在这种情况下，包装在对位膜，并储存于4℃以备后用。不推荐使用存储超过6小时长的ECM会干出。 </li></ol> 4.建立器官培养的注:此步骤介绍了如何将蜕膜器官培养和绒毛器官培养在不同的转孔。组织不接触。有兴趣在共培养技术的研究人员，其中蜕膜和绒毛是与彼此直接接触，可以参考现有文献9,10。 <ol><li>在每次冲洗之间1000 XG在收集媒体和离心冲洗标本的两倍。转移组织到无菌培养皿中，并发生在解剖显微镜的阶段。保持6孔板上相邻的显微镜冰。 </li><li>微解剖组织，用无菌施普林格解剖剪刀和镊子，建立2-3毫米绒毛器官培养秒（ 图2A）。 注:绒毛器官培养小，良好的血管"树"有2个或3个分支，带有显着外滋养细胞（EVT）电池列11。它利用来自&lt;9周头三个月只标本绒毛组织，如绒毛外滋养细胞侵入ECM最为显着这些年轻的13岁是很重要的。 </li><li>与著名的EVT细胞列选择良好的血管绒毛。 EVT单元列被可视化为球形，"模糊"，"蓬松"端部（参照图2A中箭头）。 </li><li>使用无菌镊子对3或4绒毛状树传送到每个Transwell小。调整分支所以树被平放之上ECM和独立成群的分支。 </li><li>微解剖蜕膜组织用无菌施普林格解剖剪刀和镊子，建立3 mm 3的蜕膜器官培养物（ 图3A）。 注:样品含有两种DIStinct类型蜕膜，蜕膜和顶叶蜕膜capsularis（左，右分别组织碎片， 图3A）。 </li><li>用剪刀修剪创建3 mm 3的蜕膜碎片的顶叶。蜕膜capsularis不用于这些培养。使用镊子逗走任何凝结母体血液。 </li><li>使用无菌镊子传输3-4件蜕膜到每个转孔。 </li><li>加入收藏媒体1毫升到井底部。在37℃下在5％CO 2孵育过夜板</li><li>进行实验，其中以模仿正常前三个月的胎盘氧张力，维持在相对 ​​缺氧（3％O 2），绒毛器官培养是很重要的。 注:实验室选项包括小型孵化缺氧室适合现有的内部孵化器，或引入外部N 2气体以降低氧浓度的三气培养箱。 </li><li>加入1 ml绒毛或蜕膜介质（ 标签的乐1）在培养12-16小时的transwell顶的。 注:在培养的第过夜期间缺乏媒体允许外滋养细胞的列侵入（绒毛状培养），和用于组织（包括绒毛和蜕膜）成为牢固地埋入ECM中。根据我们的经验，蜕膜器官文化保持活力72小时和绒毛器官培养96小时。我们建议属于这一期限内的实验端点。 见表1绒毛和蜕膜中的成分。绒毛状介质具有胎牛血清的高百分比，而蜕膜培养基补充有妊娠激素（孕酮，17β雌二醇），该保持蜕膜状态（ 见表1）。 </li></ol> 5.编写的L.单核细胞培养注:有关L.的长期存储详细的协议单核细胞 ，以及文化和THI增长脑心脏浸液（BHI）肉汤和琼脂小号生物体请参阅Wang等人 14 <ol><li>选择一个单一的L.从挑染BHI琼脂平板上菌落单核细胞接种和3毫升BHI肉汤。 </li><li>孵育L.单核细胞培养过夜（16小时），在一个倾斜的位置，在30℃下。 注意:这些文化条件允许细菌生长，达到稳定期L.菌在30℃下，这增加了宿主细胞侵袭15鞭毛。 </li></ol> 6.感染L.器官培养的单核细胞增生 <ol><li>温暖的PBS和绒毛或蜕膜中（ 表1）至37℃。 </li><li>使用无菌镊子除去任何器官培养片未侵入的ECM，并因此在井浮动。 </li><li>小心吸从透孔的底部媒体。轻轻吹打1毫升战争冲洗上下转孔两次中号PBS为好，小心地之间进行抽吸英寸轻轻吸取1毫升无抗生素的绒毛状或蜕膜介质的上，下的transwell。要小心，不要打扰器官培养。 </li><li>让组织在无抗生素的培养基孵育至少一个小时，以确保抗生素已被删除。 注:在固定相L.密度菌培养为每毫升约1×10 9个菌落形成单位（CFU）。用于感染器官培养的细菌数应根据经验确定，以满足实验需要。 </li><li>对于野生型L.菌等感染常规实验表明，强大的入侵与（1毫升每口井媒体4×10 7 李斯特菌 ）1:50稀释过夜。在重悬不含抗生素的绒毛或蜕膜中细菌的适当数量。 </li><li>从转孔顶吸媒体。轻轻地加入1毫升接种。在37孵育板℃，在5％CO 2，以允许细菌侵入。 </li><li>通过从上和下转孔抽吸培养基除去细胞外细菌。用温水冲洗PBS两次井。加入1毫升的绒毛或蜕膜媒体辅以50微克毫升-1庆大霉素。在5％CO 2在37℃下孵育板。 注:L。菌等是一种兼胞内细菌，可在细胞外培养基中生长，生长的细胞的内部，并从细胞至细胞传播而无需访问外空间。庆大霉素对细胞外L的杀菌作用菌 ，并且因此允许细胞内L的测量单核细胞的生长，并通过器官培养16传播。 注:可重复L.绒毛和蜕膜器官培养的菌感染5小时孵育时间发生。此外庆大霉素之前孵育时间可以被修改根据实验需要和从头机体ility侵入组织。为了更好地了解解剖部位最易受感染，更短的温育时间可以使用2。 </li><li>保持在5％CO 2在37℃下板在实验的持续时间。在每天的变化以及全媒体。 注:根据我们的经验，L.单核细胞 -感染蜕膜器官培养保持活力48小时和绒毛器官培养72小时。 </li></ol> 7.机关收获文化组织病理学<ol><li>通过在30ml PBS稀释10毫升16％的股票安瓿准备4％多聚甲醛的全新解决方案。商店4％多聚甲醛在4℃，避光，并在一周内使用。带给在使用前室温。 </li><li>从上下的transwell抽吸介质。用PBS在室温下轻轻漂洗孔两次。轻轻加入1ml 4％多聚甲醛到顶部Transwell小到完全覆盖器官培养。 </li><li>孵化在4％多聚甲醛在室温下20分钟。避免潜伏期较长时间，因为这可能会导致组织的过度固定。 </li><li>抽吸的多聚甲醛和冲洗孔用PBS两次在室温下。埋置在低温恒温器华侨城中等，冻结和部分固定器官培养（"固定冻结"）。一般来说，固定冷冻组织更容易比部分冷冻组织，恕不另行固定。 注:有关OCT包埋和冰冻切片完整的协议，请从公布的协议17,18访问相关章节。根据实验的需要和可用的实验室设备和存储，组织可以改为石蜡包埋并切片上的切片19。 </li><li>进行常规苏木HE染色，免疫，或免疫组织化学18,20,21进一步准备组织切片。 </li></ol>

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The authors have nothing to disclose.

近交系，转基因和基因敲除小鼠品系可在实验系统起到有力测试机制。然而，尽管芯遗传物质的一般保护，小鼠和人类的功能基因组展示的调节元件23显著差异。这并不奇怪，因此，在动物模型中有希望的临床前研究有时无法在人类患者中概括。胎盘显示非常高的种间差异，从而使较少的动物模型则非常适合人类疾病24的研究。双方承认在人类和小鼠免疫学25显着的差异，而在胎盘解剖明显分歧演变，谨慎的做法是考虑使用的实验研究人类妊娠组织的体外器官培养的。 的描述，照片以及视频教学在这个协议上的指示研究人员如何建立对ECM-涂透孔组织培养插入绒毛及蜕膜器官培养。这种技术的优点包括显微解剖的相对简单性和机械支承的transwell系统，特别是当与其他方法，如3D-嵌入式矩阵或器官切片培养。上膜支撑的器官培养的悬浮液允许在所有的组织表面物质交换和生存力被保持短期培养（96小时为绒毛状培养，72小时为蜕膜培养物）。本技术允许在组织研究人类胎盘生物学水平，不可否认超过单层细胞培养模型生物相关的。 在这个协议中最关键的一步是合适的绒毛及蜕膜碎片器官培养显微解剖。组织的最佳片照相证实（ 图2A和图3A），以帮助研究人员为谁妊娠标本可视化是新的。选择绒毛状树木的枝终止在EVT列，这些细胞将迁移到ECM和帮助锚定的绒毛的膜是特别重要的。对于这两种类型的器官培养的是有帮助的过程中，吹打在仔细和从容地媒体的变化，以尽量减少干扰，组织。 该技术提出了只有很少的困难。有时，标本展示污染。污染通常是细菌（偶尔聚微生物），以及只在培养1-2天后变得明显。一旦污染物被观察到的，漂白应适用，将培养丢弃，并且组织培养箱灭菌，以防止一个长期的问题。有几种可能的方式污染可能出现:在子宫内感染，手术过程中，试样收获/处理期间，或在器官培养维护。收获和加工步骤是最可能的时间和将要引入的污染的地方。因此，为减少的时间收集和显微解剖期间检体被操纵的量是重要的。这将减少样品暴露于周围环境，非无菌的环境。根据实验室设置中，解剖显微镜可以位于无菌组织培养罩内。 器官培养的组织病理学分析感染后与医学相关的病原体L.菌 ，在此显示作为协议的一个可能的应用。感染后的产量新的见解人类宿主对病原体细菌和宿主免疫细胞的免疫荧光定位。蜕膜器官培养可以作为一种补充体外实验方法检测在小鼠体内的感染，包括显示在小鼠巨噬细胞蜕膜防御功能缺陷26,27耐人寻味报告生成的数据。此外，人类组织一个培养物可以在混合感染策略中使用，如L的几个等基因的菌株组成的有竞争力的接种物菌等 。器官培养的有竞争力的感染是测试人类宿主中的毒力因子的相关性的灵敏方法。这种方法的一个制约因素是组织活力，这是仅限于短期培养（96小时为绒毛培养，72小时为蜕膜文化）。这是理想的感染快速增长的微生物如L.菌 ，但更长培养可能需要对于需要更多的时间来建立，并通过组织传播的病原体。 为了减少妊娠并发症的全球负担，研究必须着重了解人类母胎界面的病理生理学。细菌，真菌，以及从母亲跨越给胎儿造成毁灭性的怀孕和胎儿感染等并发症病毒病原体。受控实验室 ANI 体内感染马尔斯通常被视为黄金标准解决病原体如何殖民和器官28日期间中转。由于胎盘解剖整个哺乳动物显着变化，这是最重要的，把人权组织为研究策略。人类绒毛及蜕膜机关文化是高度相关的模型系统来研究宿主 - 病原体相互作用。为了研究这一重要但知之甚少的器官，研究人员可以利用丰富否则丢弃的人胎盘及蜕膜标本的优势，利用器官培养策略，如这里所描述。

感染和炎症在母胎接口表示妇女和儿童发病率和死亡率的主要来源。理解病原体如何感染这些组织为的新策略的发展，以预防和治疗疾病，例如早产和胎儿死亡的关键。然而，母胎界面的高变异物种间实验研究复杂化。此外，大多数微生物病原体显示宿主特异性，因此许多动物模型不能完全概括人类传染病。某些生物（ 流感嗜血杆菌 ， 伤寒沙门氏菌 ， 霍乱弧菌 ，以及众多的其他人）是在人类中严格致病的，而另一些，如李斯特菌，显示宿主特异性1的多个中间级。宿主特异性已被记录在发病机制中的许多方面，包括定植2，传播3，免疫逃逸4 </sUP&gt;和养分收购5。因此，它是最重要的，选择最佳主机模型系统。 由胎儿细胞和母体血液中，胎盘是一个迅速发展超过6酝酿的过程中的器官。在最简单的方面，人胎盘被构造在母血沐浴胎儿"绒毛"树形结构。天然气和养分交换横跨称为该行的绒毛树的表面滋养胎儿的专门细胞层发生。母体子宫粘膜衬，在非妊娠雌性称为内膜，结构和功能上转换为蜕膜以容纳胎儿胎盘单元。绒毛树被固定成从锚定元件列入蜕膜迁移绒毛外滋养层细胞（EVT）子宫。母胎界面，因此，由蜕膜和胎盘。 器官培养技术这里描述已被用于检查在何处以及如何临床相关的人类病原体，包括L。 monocytogene S和弓形虫 ，穿过胎盘屏障2,7。这些体外器官培养复制体内组织架构，以及对人类胎盘可以说是生理上高度相关的模型系统。其他养殖技术包括全器官外植体，器官切片和组织或干细胞组织体嵌入的3D矩阵。有关这些选项的详细信息，请参阅进行全面检讨最近8。请注意，这个协议是蜕膜和胎盘绒毛的独立的文化。用于研究的胎盘细胞和蜕膜细胞之间的相互作用，共培养技术是优选的。我们是指有兴趣的读者到先前描述的绒毛-蜕膜共培养技术，通过研究人员在研究滋养层-介导的蜕膜血管重塑9-11使用。

<table><tbody><tr><td>Sterilization pouches</td><td>Fisher Scientific</td><td>01-812-54</td><td>For autoclaving individual dissecting tools</td></tr><tr><td>Fine mesh strainer</td><td>Cuisinart (Amazon.com)</td><td>NA</td><td>Wrap completely in aluminum foil and autoclave prior to tissue collection.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Carboy with spigot</td><td>Fisher Scientific</td><td>03-007-647</td><td>For large volume preparation of Wash buffer.</td></tr><tr><td>Ice packs</td><td>Nortech labs</td><td>GB8818</td><td>These do not have to be purchased, rather they can be recycled/reused from any routine laboratory shipment that includes them in the packaging.</td></tr><tr><td>70% Ethanol</td><td>VWR</td><td>V1001</td><td>70% solution made by adding dH&lt;sub&gt;2&lt;/sub&gt;0 to 190 or 200 proof research grade alcohol.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>10% Bleach</td><td>Waxie Sanitary Supply</td><td>CLO 30966</td><td>10% solution made by adding dH&lt;sub&gt;2&lt;/sub&gt;0.</td></tr><tr><td>Light Box</td><td>Litebox Lumina (dickblick.com)</td><td>55305-2009&amp;nbsp;</td><td>Note replacement bulbs also purchased on Blick (55305.0100)</td></tr><tr><td>Micro dissecting forceps&amp;nbsp;</td><td>Stoelting&amp;nbsp;</td><td>52102-43</td><td>4 inches, 1 x 2 x 0.5 mm&lt;sup&gt;3&lt;/sup&gt;, Slight Curve</td></tr><tr><td>Micro dissecting forceps</td><td>Stoelting</td><td>52102-06</td><td>4 inches, Straight Fine, Sharp</td></tr><tr><td>Micro dissecting vannas spring scissors</td><td>Stoelting&amp;nbsp;</td><td>52130-01P</td><td>McPherson-Vannas Spring Scissors, 8.5 cm, 0.33 Tip, Slight Curve</td></tr><tr><td>Dissecting microscope</td><td>Leica Microsystems</td><td>MZ16 or M60</td><td>We have had success with the listed models.&amp;nbsp; External gooseneck flexible light sources are helpful but not necessary.</td></tr><tr><td>50 ml conical tubes</td><td>Sigma-Aldrich (Corning)</td><td>CLS4558</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco (ThermoFisher Scientific)</td><td>10010023</td><td>We purchase from our university Tissue Culture Core facility, alternate options such as this are available.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>10x Phosphate Buffered Saline</td><td>Teknova</td><td>P0195</td><td>For preparation of Wash buffer we use 10x PBS</td></tr><tr><td>DMEM/F-12 nutrient mixture (Ham&#039;s) with GlutaMAX</td><td>Gibco (Life Technologies)</td><td>10565-018</td><td>We purchase this specific media formulation, containing 2.438 g/L sodium bicarbonate, 55 mg/L sodium pyruvate, and 4.5 g/L glucose</td></tr><tr><td>Gentamicin</td><td>Thermofisher Scientific</td><td>15710072</td><td>1, 000x stock. Recommend to prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (ThermoFisher Scientific)</td><td>15140-122</td><td>100x stock.&amp;nbsp; Recommend to prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Amphotericin B</td><td>Gibco (ThermoFisher Scientific)</td><td>15290-018</td><td>500x stock.&amp;nbsp; Recommend to prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>A 1 mM stock solution is made by reconstituting 15.7 mg progesterone powder in 15.7 ml Ethanol and adding 34.3 ml PBS. Solution is sterile filtered, aliquoted, and stored at -20 &amp;deg;C.</td></tr><tr><td>17&amp;beta;-estradiol</td><td>Sigma-Aldrich</td><td>E2758</td><td>A 10 &amp;mu;M stock solution is made by reconstituting 13.5 mg estradiol powder in 10 ml ethanol and adding 40 ml PBS. Solution is sterile filtered, aliquoted, and stored at -20 &amp;deg;C.</td></tr><tr><td>Bottle-top vaccum filter (0.22 &amp;mu;m)</td><td>Sigma-Aldrich (Corning)</td><td>CLS430769</td><td>For sterile filtration of Collection media after preparation</td></tr><tr><td>6-well tissue culture plate</td><td>BD Falcon</td><td>353224</td><td>Polystyrene, Tissue culture treated</td></tr><tr><td>6-well transwells&amp;nbsp;</td><td>Millipore</td><td>PICM03050</td><td>Insert - 30 mm diameter, 0.4 &amp;mu;m pore size hydrophilic PTFE membrane</td></tr><tr><td>Extracellular Matrix (for example, Matrigel Matrix)</td><td>BD Biosciences</td><td>354234</td><td>We have utilized Matrigel Matrix in our studies. It is a solid at room temperature and at -20 &amp;deg;C. Avoid repeat freeze/thawing. Thaw bottle to viscous solution at 4 &amp;deg;C, and prepare ~ 300 &amp;mu;L aliquots in the cold room with chilled pipette tips. Store aliquots at -20 &amp;deg;C.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Paraformaldehyde, 16% w/v aqueous solution&amp;nbsp;</td><td>Alfa Aesar</td><td>30525-89-4</td><td>For tissue fixation, a fresh preparation of 4% paraformaldehyde is made by diluting this stock in PBS.</td></tr><tr><td>Tissue culture incubator, maintained at 37 &amp;deg;C, 5% CO2, 3% oxygen (optional for villous organ cultures)</td><td></td><td></td><td>For some experiments, hypoxia may be preferred.&amp;nbsp; This can be established multiple ways, including addition of exogenous nitrogen via gas cylinder, Tygon tubing, and a regulator.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Bench top centrifuge</td><td></td><td></td><td></td></tr></tbody></table>

human placental and decidual organ cultures to study infections at the maternal-fetal interface

胎盘显示出很大程度的种间解剖变异。为了更好地理解生物学和人类胎盘的病理生理，当务之急是要设计出使用人体细胞组织实验。器官培养的优点是三维（3D）的维护结构组织和细胞外基质。这里所描述的方法的目标是成功地建立体外人体妊娠组织器官培养物和72-96小时他们健康培养维修。协议细节的研究，同意，胎盘及蜕膜标本从操作套件新鲜立即处理。这些丰富的标本，否则将被丢弃。对这些样品，包括关于如何选择适当的组织，以建立三维器官培养形态学细节的无菌采集的详细说明，提供了一种。胎盘绒毛及蜕膜组织被显微切割为2-3毫米的3块和矩阵林立的transwell过滤器分开放置，并培养了好几天。绒毛及蜕膜器官培养是非常适合人类宿主 - 病原体相互作用的研究。相对于其他的模式生物，这些人类文化是特别有利的，以检查该证明宿主特异性的可变图案的病原体感染的机制。作为一个例子，我们证明胎盘和蜕膜器官培养的感染与临床相关，兼胞内细菌病原体李斯特菌。

图1（来自泽利多维奇＆Bakardjiev 22修改）图有关的胎盘和子宫解剖结构。 图2示出了代表毛和胎盘绒毛的组织学图像，而图3显示蜕膜组织。 该协议第一次描述了在医院或诊所的新鲜胎盘及蜕膜组织的集合。所收集的样本是胎儿胎盘（绒毛）与母体子宫组分（蜕膜）的混合物中。用含有广谱抗生素和抗真菌剂缓冲器漂洗后，将整个样品是由眼睛使用灯箱检查。绒毛和蜕膜被放置在单独的管中并输送在冰上至实验室。在实验室中，在解剖显微镜来欣赏健康组织的细形态学特征。代表照片绒毛（ 图2A）及蜕膜 （ 图3A），该被获得与两个外部鹅颈光源解剖显微镜示作参考。 绒毛器官培养从孕早期标本产生。文化包含有2-6支小型终端绒毛树木。它解剖绒毛状分支的终止外滋养细胞柱和是良好的血管化是非常重要的。这些特征被视为"蓬松"或"模糊"分支端（箭头），而微弱的粉红色到红色线性色调，通过在图2A中的左侧的树的分支的课程。相比之下，血管和绒毛外滋养层细胞不容易显现对这一形象的权利，这将是次优的文化树。过培养的第一天，绒毛外滋养细胞迁移到的ECM，叔HUS锚定绒毛树插入透孔矩阵。 蜕膜器官培养是从第一和早期孕中期标本产生。整个子宫内壁被植入后转化为蜕膜很快。更具体地说，底蜕膜定义了子宫粘膜下层直接胎盘/着床部位，蜕膜capsularis定义黏膜覆盖胎儿和蜕膜顶叶是指子宫内膜的剩余部分。在解剖显微镜下的可视化显示，早期妊娠标本由主要的蜕膜顶叶和蜕膜capsularis（分别左右组织片段，在图3A）。该蜕膜capsularis容易被其薄，膜的性质区分。对于器官培养，蜕膜顶叶被调整到3毫米3 微解剖剪刀和阿德片段租金血块是用镊子取出。 以下培养过夜后，组织被感染L.菌等 。上述感染协议是基于用于研究细胞内生长和兼性胞内细菌16的传播的庆大霉素保护测定。在不含抗生素的培养基器官培养物孵育L.单核细胞 5小时。培养物用无菌PBS洗涤，以从培养基中除去细菌。此后，庆大霉素加到消除细胞外生物。从我们的实验室在此之前文献中使用免疫荧光和共聚焦显微镜感染后2来确定不同时间点细菌的组织定位。器官培养物漂洗在PBS，固定在4％多聚甲醛，和任一冷冻包埋于OCT中，储存在-80℃，或石蜡包埋和在室温下储存。组织切片可站INED对于免疫荧光（IF）或细菌的免疫组织化学（IHC）分析和真核细胞的定位。 图2B是一个典型的IF在感染后72小时从8.3周绒毛状器官培养制备的冰冻切片的图像。请注意，在EVTs沉重的负担细菌（绿色荧光）在绒毛分支的末端，和树衬合体滋养细胞（红色荧光层）中的相对缺乏细菌。在以往的工作中，我们使用了类似的IF染色本地化L.单核细胞在感染后的不同时间点的绒毛器官培养。超过72小时，感染扩散，从EVT为子合体滋养细胞和底层绒毛间质。值得注意的是，synyctiotrophoblast层是感染2抗性。 H＆E染色石蜡包埋的蜕膜组织切片的代表性光镜图片来自14.3周specime准备纳秒示于图3B和3C。此ECM涂覆的transwell膜埋入到显示原位器官培养（ 图3B）。图像显示上皮衬里腺体（星号）和内皮衬里血管（钻石）蜕膜基质细胞室中不均匀的位置。此外，还有在可变密度散布在蜕膜间质母体免疫细胞。 IHC和IF染色可以用于确定细菌显微解剖定位，相对于特定的驻留的免疫细胞。作为一个例子，CD14 +巨噬细胞（绿色荧光）定位于脉管系统的衬里 ​​和分散在基质（ 图3D）内，而L的大的聚集体菌 （红色荧光）在感染后48小时（ 图3D）中的蜕膜间质主要观察到。 <table border="1" fo:keep-together.within-page="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 洗涤液 </td><td> 集合中 </td><td> 中等绒毛 </td><td> 蜕膜中 </td></tr><tr><td> PBS </td><td> DMEM / F-12含有Glutamax </td><td> DMEM / F-12含有Glutamax </td><td> DMEM / F-12含有Glutamax </td></tr><tr><td>青霉素100IU /毫升</td><td>胎牛血清2.5％ </td><td>胎牛血清20％ </td><td>胎牛血清2.5％ </td></tr><tr><td>链霉素100微克/毫升</td><td>青霉素100IU /毫升</td><td>青霉素100IU /毫升</td><td> 17β雌二醇300微克/毫升</td></tr><tr><td>庆大霉素50μg/ ml的</td><td>链霉素100微克/毫升</td><td>链霉素100微克/毫升</td><td>黄体酮20毫微克/毫升</td></tr><tr><td>两性霉素B 1.25微克/毫升</td><td>庆大霉素50μg/ ml的</td><td></td><td></td></tr><tr><td> &lt;<html> / TD&gt; <td>两性霉素B 1.25微克/毫升</td><td></td><td></td></tr></tbody></table> 表1:媒体食谱组件和浓度制备洗涤缓冲液，收集培养基，绒毛介质，和蜕膜培养基。 <img alt="图1" src="/files/ftp_upload/54237/54237fig1.jpg" /> 图1.胎盘结构（ 一 ）胎儿-胎盘单位在产妇子宫结构，（B）框在扩大（A）强调，胎儿滋养层（T）内衬胎盘绒毛沐浴在母亲的血液和锚入蜕膜绒毛外滋养层细胞（EVT）。绒毛中都包含胎儿的血管（FV），成纤维细胞和胎儿的巨噬细胞。从泽利多维奇＆Bakardjiev 22修改。<html> "&gt;请点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54237/54237fig2.jpg" /> 图2: 绒毛器官培养-代表肉眼和显微镜图像 （A）6周的胎龄两个终端绒毛状的树木，在解剖显微镜下观察为。请注意"蓬松"结束（箭头）和突出的胎儿血管通过，使这片适合器官培养左侧的树枝窃喜。 （B）器官培养（胎龄8.3周）72小时后感染的免疫荧光显微镜李斯特菌，突出重细菌负荷[绿色]在绒毛外滋养。 DAPI显示为蓝色，红色βHCG+合体。比例尺= 1毫米（A），250微米（B）。7 / 54237fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54237/54237fig3.jpg" /> 图 3: 蜕膜器官培养-代表肉眼和显微镜图像 （A）蜕膜顶叶[左]及蜕膜capsularis [右]在孕龄6周，在解剖显微镜下观察为。 14.3周孕周蜕膜组织器官顶叶文化（B）H＆E染色的部分显示了整个间质蜕膜组织不均匀腺体和血管。在（钻石，◆）和（星号*）突出代表容器密封，分别为。在下边布朗线转孔膜边缘。 （C），H＆E染色的14.3周孕周蜕膜顶叶部分器官培养在更高的放大倍率ðemonstrates嵌入在蜕膜间质肌壁螺旋小动脉。母体免疫细胞小暗圆形核，并在整个基质不规则地分布。 （D）器官培养48小时后感染L的免疫荧光显微菌 ，细菌凸显大区[红色]蜕膜间质，和产妇CD14 +巨噬细胞[绿色]衬血管迂曲空间和散落在基质。比例尺= 1毫米（A），250微米（B），50微米（C和D）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54237/54237fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface at JoVE.com

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

被描述为建立原代人胎盘（绒毛）及蜕膜器官文化的简单方法。绒毛及蜕膜器官培养是在人类母胎界面发病机制研究的宝贵工具。感染的细胞内兼细菌李斯特菌是证明。

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

human-placental-decidual-organ-cultures-to-study-infections-at

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.2K 次观看.  Benioff Children’s Hospital. 被描述为建立原代人胎盘（绒毛）及蜕膜器官文化的简单方法。绒毛及蜕膜器官培养是在人类母胎界面发病机制研究的宝贵工具。感染的细胞内兼细菌李斯特菌是证明。 

视频: Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

科研

发育生物学

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

生物工程

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

免疫与感染

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

人类羊膜不同解剖区域的皮下细胞体外培养

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

使用基于流式的方法改善离体胎盘外植体培养

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

作者聚焦:通过胎盘组织中的单细胞测序和空间组织学推进研究 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

人胎盘及蜕膜器官培养研究在母胎界面感染

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此视频中的章节

从人胎盘组织源分离和间充质干扩张/基质细胞

通过胎盘的外来物质和纳米材料的运输率测定

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

从人类母胎界面分离白细胞

从小鼠组织在母胎界面分离白细胞

人类羊膜不同解剖区域的皮下细胞体外培养

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

离体胎盘外植体流动培养 - 模拟子宫内的动态条件

分离小鼠原代滋养细胞及滋养细胞侵袭实验