Supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (no. 262011-2009) to CV and March of Dimes Social and Behavioral Sciences Research grant (#12-FY12-179) to CV and JTS; by studentships to LSF from the Minist&#232;re de l'&#233;ducation, de l'Enseignement sup&#233;rieurs et de la recherche (MEESR)-Fonds de recherche du Qu&#233;bec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the R&#233;seau Qu&#233;b&#233;cois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Sant&#233;, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cient&#236;fico e Tecnol&#242;gico (CNPq) and the Programme de bourses d'excellence pour &#233;tudiants &#233;trangers MEESR-FRQNT.

从简单怀孕的密友 - 圣 - 吕克医院，蒙特利尔，加拿大自然阴道分娩后立即获得胎盘，有知情患者同意和伦理委员会（CHUM圣 - 吕克医院和INRS学院阿尔芒 - Frappier批准，拉瓦尔，QC，加拿大）。 绒毛细胞滋养层细胞1.分离和纯化<ol><li>解决方案和媒体<ol><li>通过补充准备传输介质贝科改良Eagle培养基高糖（DMEM-HG）用1％ 体积/体积的抗生素（10,000单位/ ml青霉素G，100毫克/毫升硫酸链霉素）和储存在4℃。 </li><li>通过用10％ 体积/体积的胎牛血清（FBS），25mM的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），1％ 体积/体积的抗生素（10,000单位/补充的DMEM-HG制备原代培养介质毫升青霉素G，100mg / ml的链霉素硫酸盐），并存储在4℃。温暖的媒体至37℃之前使用。 </li><li>制备4升的盐水溶液（0.9％重量/体积的氯化钠）。 </li><li>准备加入的25mM HEPES至1x HBSS（pH 7.4）中改性Hank氏平衡盐溶液（HBSS）中。 </li><li>制备的消化溶液新鲜，四瓶具有修饰的HBSS（在1.1.4的方法制备），硫酸镁（MgSO 4上），氯化钙（ 氯化钙 ），胰蛋白酶，脱氧核糖核酸酶IV（DNA酶IV）和1％ 体积/体积的抗生素（ 10,000单位/ ml的青霉素G，100毫克/ ml硫酸链霉素），如表1所示。 </li><li>准备密度离心媒体梯度<ol><li>通过补充密度离心介质，用10％ 体积/体积的HBSS 10倍制备密度离心介质溶液。 </li><li>制备具有密度离心介质溶液和改性的HBSS 14测定管，如表2所述。 </li><li>大力增加其内容的梯度之前每个混合密度的解决方案（步骤1.1.6.2）。平缓用蠕动泵（1毫升/分钟）中，加入浓度溶液到50ml玻璃离心管中，以最高浓度（70％）开始。通过排放靠在管壁溶液保持梯度的每层的适当分离，避免液滴。 <ol><li>在没有蠕动泵，轻轻敷层采用巴氏吸管。 </li></ol></li></ol></li><li>准备通过补充运行缓冲液最终运行缓冲液（见材料的数据表），用2％ 体积/体积的抗生素/抗 ​​真菌剂（10,000单位/ ml青霉素G，100毫克/ ml硫酸链霉素）。保存在4℃。 </li></ol></li><li>绒毛细胞滋养层隔离 注意:使用无菌手术器械，玻璃仪器，移液管，烧瓶等 。 <ol><li>上绒毛细胞滋养层隔离的当天，在37℃水浴中，温消化溶液（从步骤1.1.5）和70的FBS毫升。注意:使用50毫升FBS的中断消化和剩下的20毫升，可置于冰上解冻一次冷冻工序（1.2.22）。 </li><li>分娩后，使胎盘在冰冷的传输介质（来自步骤1.1.1）实验室尽快（小于1小时）。 </li><li>放弃传输介质和胎盘血液体垃圾箱。称取胎盘，冷盐水浸泡。 </li><li>测量和分析以下特点:脐带的长度;脐带定位;胎盘的长度，宽度，形状（椭圆形，盘状）;膜的颜色;子叶结构的病症。注:数据在结果部分提出。 </li><li> （用线缠绕的附加​​1厘米圈半径即在其基）剪断脐带的旁边插入胎盘。浸泡在300毫升病理组织固定液（福尔马林10％）供以后组织学分析的。 </li><li>切整个胎盘进入5×5×5厘米的立方体。彻底清洗（4次X〜1分钟）生理盐水溶胶ution（0.9％）以除去血细胞，直到盐溶液是明确的。弃去冲洗液。 </li><li>在手表的玻璃，取出胎盘胎膜切末组织去除血管和钙化。用钳子紧紧握住血管和删除使用剪刀梅岑鲍姆的背部组织。 <ol><li>放置剁碎胎盘在一个布氏漏斗。用有约100ml生理盐水缓冲液漂洗。继续切碎，直到30 - 35克剁碎组织获得（使用塑料称量船和规模）。如果需要的话，肉的胎盘剩下来获得多达三个额外的30 - 35克准备。在此期间，把组织糜在称量舟上冰上。 注意:此步骤将花费大约45分钟至1小时。 </li></ol></li><li> 35克剁碎胎盘组织到胰蛋白酶瓶 - 30添加。转让150毫升准备消化液1（ 见表1）的胰蛋白酶消化烧瓶中拌匀。 </li><li>放置胰蛋白酶消化烧瓶中一个在不超过50个循环/分钟的速度振摇水浴30分钟并手动混合胰蛋白酶烧瓶每5分钟均匀消化。 </li><li>在第一消化的末端，取出从水浴的胰蛋白酶烧瓶和其倾斜（45°）为1分钟以沉淀胎盘组织。用10毫升无菌吸管，删除和丢弃约80ml上清液。避免吸组织。 </li><li>将100毫升消解液2（ 表1）的胰蛋白酶瓶;拌匀，重复步骤1.2.9。 </li><li>在第二消化的末端，取出从水浴的胰蛋白酶烧瓶和其倾斜45°1分钟。用10毫升无菌移液管，取出80毫升上清液和轻轻地用细胞过滤网（100微米目）转移到一个离心管中。传送过滤的上清液含有每离心管充满时2毫升FBS的烧杯中。 </li><li>作为所述执行第三消化使用75毫升消解液3.在平行的第二个消化（1.2.11和1.2.12），执行步骤1.2.15到1.2.16.1消化2。 </li><li>准确执行第四消化作为使用消化溶液4（75毫升）中的第三消化，但收集上清的最大量。执行步骤1.2.15到1.2.16.1并行消化3，最后消化4。 </li><li>等分试样（来自消化2，3和4）将上清液进13.5毫升部分，每个部分为一15ml离心管中。随着22.8厘米长的玻璃巴斯德吸管，轻轻地，慢慢地放置1.5毫升FBS在每个管的底部，以创建一个单独的层。不要混合FBS和上清液。离心管不带制动器20分钟，在室温1250 XG。 注意:离心后，4层是可见在管， 如图1胰蛋白酶和滋养细胞的分离避免了过多的细胞消化。 </li><li>用真空泵，aspir吃并丢弃上清液（消化溶液）和FBS层，包括两个层之间的白色膜。重悬沉淀（滋养细胞和红血细胞层）用1毫升温细胞培养基（步骤1.1.2;无FBS和没有HEPES）。 <ol><li>收集所有的管子悬浮细胞，并在1管将它们结合起来。让该管在室温下放置直到所有消化的末端。注:所有的消化之后，通常的产量悬浮细胞（消化每一个）3管。 </li></ol></li><li>补足体积至15ml用温细胞培养基。离心在1250 xg离心在室温下10分钟。除去用真空泵上清。避免吸沉淀。 </li><li>轻轻悬浮用1毫升温细胞培养基从3管得到的粒料。他们集中在1管的内容。要获得8毫升，完成与温暖细胞培养基的体积。 </li><li>轻轻在分离层毕业生细胞悬液ient用巴氏吸管。没有制动离心30分钟，在室温下507 xg离心。 </li><li>离心后，确定在与背面照明梯度细胞的不同的层。找到包含滋养层和40之间污染细胞层 - 密度离心培养基的50％。用真空泵，除去上层（&gt; 50％）。 </li><li>收集位于的用巴氏吸管兴趣层细胞，并将它们传送到一50ml离心管中。补足体积至50ml用细胞培养基。在室温下以1250 xg离心离心10分钟。 </li><li>在无菌条件下，弃上清，重悬用20ml的FBS的颗粒和计数使用血球细胞的数目。在冰，加2.22毫升无菌二甲基亚砜（DMSO），并通过翻转轻轻拌匀。细胞悬浮到低温小瓶等分1.5毫升，在-80℃冷冻过夜，然后转移至液氮罐中。 </li></ol></li> &lt;LI&gt;净化滋养层<ol><li>安装在漂洗和运行缓冲液和上根据制造商的说明在磁性纯化仪器新的过滤器柱。执行"清洁程序"，以干净的负1，积极的1和2阳性端口，然后根据制造商的说明每个端口下引入50ml试管。 </li><li>解冻在步骤1.2.22在37℃水浴中被冻结迅速的细胞。转移细胞到50ml管中，轻轻地用20ml冷的流动缓冲液悬浮细胞。离心反应管在450 xg离心5分钟，4℃。 </li><li>弃去上清液。重复洗涤步骤用冷运行缓冲液。使用血细胞计数器确定可行性计数细胞。重复离心（5分钟，450 xg离心在4℃）。小心取出上清液。加入1毫升含有1％ 体积/小鼠抗-HLA-ABC抗体的体积冷运行缓冲液中。在4℃孵育30分钟，mixi网纳克轻轻每隔5分钟。 </li><li>加6毫升冷运行缓冲液。离心机在450 xg离心5分钟，4℃。弃上清，重复此步骤。悬浮细胞在1ml含有10％ 体积/抗小鼠二级抗体偶联的磁珠体积冷运行缓冲液中。孵育在4℃下15分钟，轻轻混合，每5分钟。 </li><li>加6毫升冷运行缓冲液。离心机在450 xg离心5分钟，4℃。丢弃在5毫升冷磨合缓冲上清，重悬。 </li><li>使用磁净化仪器分离滋养层细胞。收集细胞在负端口，加入20毫升冷运行缓冲液。 注意:滋养层细胞不包含复杂的HLA-ABC，并因此从其它细胞类型分开，朝向负1端口。 </li><li>离心机在450 xg离心5分钟，4℃。弃去上清液并轻轻重悬细胞于20毫升温暖原培养基。全光照计数细胞GA血球确定可行性。 </li><li>板中的细胞在以下密度:0.15×10 6个细胞中/孔的96孔板，1.6×10 6个细胞中/孔24孔平板和4.5×10 6个细胞/孔在6孔板中。在37℃和5％CO 2孵育板。 </li><li>确认滋养细胞通过流动纯度16,17仪和/或免疫细胞化学18。 注意:在使用FITC缀合的单克隆抗体对细胞角蛋白-7和波形蛋白17-19测定免疫纯化的细胞的纯度。该协议是在Lanoix 等人以及详细的，2008年7月。 </li><li>至少4小时后，冲洗细胞两次用温培养基以除去未附着细胞，然后在板传送到常氧腔室，它是由8％O 2（ 见图2和第2部分）的。 </li></ol></li></ol> 2.体外感应仪常氧n和缺氧/复氧<ol><li>孵化室的操作（参见设置图2A）。 <ol><li>在层流罩，将含有无菌水在培养箱室，以避免干燥的底部培养皿;然后将先前制备的细胞培养板或烧瓶（步骤1.3.10）在腔室的上货架上。 </li><li>罩外，附加的腔室（吸入口）的气体的软管（ 图2A:图5a，b和c），以达到气体（8％O 2或0.5％O 2）的管（ 图2A:7）。打开腔室的入口和出口端口。在这一刻，该气体调节器（ 图2A:4）应该保持封闭。 </li><li>小心打开气体调节阀（ 图2A:4）。冲洗为25升的空气流4分钟/ min至完全取代空气室内部。 </li><li>冲洗室后，关闭燃气调压器，然后入口和OU腔室的tlet端口。 </li><li>拔掉流量计出口软管（ 图2A:5C）从室的入口，并放置在细胞培养恒温箱腔室在37℃。 </li><li>通过步骤2.1.3后气体1小时填充所述腔室替换当前存在于该板，烧瓶，溶解于培养基中的空气。 </li><li>重复步骤2.1.1 2.1.6其他气体组成（ 如 2％O 2早孕滋养细胞培养9）。 </li></ol></li><li>确认氧气百分比（ 图2B-C） <ol><li>为了确认氧在细胞培养基中室内部的浓度（无细胞），使用连接到一个氧适配器氧电极。氧电极连接到一个电压计。 </li><li>创建在同一溶液中的标准曲线（ 即 ，细胞培养基）通过溶液到气体与已知的氧含量（曝光例如 ，0％和21％的氧气）。读数是稳定的对于每个浓度后，引入电极到该室中的介质。 注:以所有测量在相同的深度，以避免在氧浓度10任何偏差。 </li></ol></li><li>在滋养细胞常氧和缺氧/复氧的诱导。 <ol><li>增加的主要滋养细胞到合适的细胞培养瓶，板或培养皿后，进行必要的治疗。 </li><li>并行地，腔室的内部，细胞暴露于所需的气体混合物以再现特定的条件，每24小时（ 图3）。 </li></ol></li></ol>

The authors have nothing to disclose.

在哺乳动物中，胎儿发育是直接依赖于足够胎盘功能。健康障碍的发展起源是基于的以后的生活中表现的疾病的病因可追溯到早期的发展，而且胎盘在胎儿编程30-32一种机械作用的假说。胎盘是胎儿生长发育的关键调解人:它调节养分转移，防止有害的曝光，并具有重要的内分泌功能。可重复的方法来隔离可行的主要滋养由Kliman 等的发展。在正常和异常胎盘功能6研究?...

整个人类怀孕，胎盘滋养层细胞，这是单个核干细胞，快速增殖和分化成绒毛或绒毛外滋养层细胞。绒毛外滋养细胞侵入并重塑子宫壁的螺旋动脉。绒毛细胞滋养细胞，另一方面，不断地增殖，分化和融合形成多核合体（合胞体）1。绒毛滋养细胞稳态的维持是胎儿的福祉和健康的怀孕是必不可少的。事实上，绒毛滋养细胞允许氧气和营养物质的母胎交换，并产生必要的荷尔蒙怀孕。此外，该合体是唯一的细胞类型与母体血液循环的直接接触，并提供了一​​个基本的物理和免疫障碍。因此，必须合体凋亡和替代稳态维护和AVOID胎盘病变2-5。 通过Kliman 等 6在1986年发展到初级绒毛细胞滋养细胞从人胎盘分离的技术中，允许参与绒毛滋养细胞分化的分子机制的研究导致胎盘研究了一场革命。这种古典工艺的基础上，用胰蛋白酶和DNA酶连续酶消化，其次是密度离心分离介质（胶体二氧化硅颗粒涂层由聚乙烯吡咯烷酮，或珀）是目前公认的金标准隔离绒毛细胞滋养层细胞。该技术可以通过磁免疫纯化，即从基于这些细胞的表面上的特异性抗原的差异表达非滋养层细胞分离绒毛细胞滋养层的过程进行优化。我们选择了人类白细胞抗原ABC（HLA-ABC）由于对滋养细胞MEMBRAN没有其表达Ë7,8。 胎盘是妊娠期间发生的氧气含量急剧变化的机构。在第一三个月，充氧比率是生理上非常低的（2％O 2），但增大到在第二和第三个三个月氧合温和水平（8％O 2）。 Tuuli 等人 9描述了胎盘绒毛内的滋养环境的体外再生是在氧合水平的挑战和变化甚至可能导致表型的改变。正是因此，建议采用8％的氧作为常氧模仿怀孕8,9的3个月期间胎盘绒毛中发现的氧张力。 Chen等人 10广泛的研究与在滋养层细胞培养物中的氧张力几个变量和表现出确定在细胞周围环境中的氧含量的重要性。氧在绒毛水平趋向于增加由于血管。在不断胎盘绒毛增加血流和过氧化氢 ​​的水平（一种丰富的活性氧）是控制血管11,12的重要信号。在妊娠并发症，缺乏血管生成的产生缺氧，更重要的是，氧的间歇性变化（称为缺氧/复氧）。这些条件导致氧化应激的异常增加，这影响胎盘和胎儿的生存能力13,14。即滋养层细胞缺氧/复氧发作期间在体内经历的改变可以在体外模仿如下:绒毛细胞滋养细胞中含氧量正常的条件（8％O 2），直到它们分化 ​​成合体下保持。然后将它们进行4小时低氧条件（0.5％O 2），其次是常氧（复氧）的另外的18小时。使用这种缺氧/复氧的做法，滋养细胞前hibit解除管制的氧化还原状态和增加的固有凋亡8的水平，正如在某些妊娠并发症被观察到。因此，这是一个有用的体外模型来评估，以打击胎盘缺氧/复氧关联妊娠并发症的新的预防和治疗的方法。 胎盘细胞产生的褪黑激素，其具有几个重要的功能，如要避免氧化应激和胎盘功能不全15的能力。这里，我们目前使用在分子，细胞和功能8级证明褪黑激素在胎盘滋养层细胞的保护作用的实验方法和细胞模型。

<table><tbody><tr><td>Curved Metzenbaum Scissors</td><td>Shandon</td><td>9212</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Splinter Forceps Fine 41/2 in</td><td>Fisherbrand</td><td>13-812-42</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Scissors 4.5 Str Dissection</td><td>Fisherbrand</td><td>08-940</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Gauze Sponge 10 cm x 10 cm</td><td>Cardinal Health</td><td>361020733</td><td></td></tr><tr><td>Oblong Glass Baking Dish</td><td>Pyrex</td><td>1105397</td><td>Glassware (2.8 L)</td></tr><tr><td>Funnel Buchner</td><td>Coorstek Inc</td><td>10-356E</td><td>Glassware (114 mm diameter)</td></tr><tr><td>Watch Glass&amp;nbsp;</td><td>pyrex</td><td>9985100EMD</td><td>Glassware</td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td>histological tissue fixative solution</td></tr><tr><td>Trypsinizing Flasks</td><td>Wheaton</td><td>355395</td><td>Glassware (1 unit)</td></tr><tr><td>Disposable Culture Tubes</td><td>Kimble</td><td>73750-13100</td><td>Glassware</td></tr><tr><td>Borosilicate Glass Pasteur Pipet (22.8 cm)</td><td>Fisherbrand</td><td>K63B1367820C</td><td>Glassware</td></tr><tr><td>250 ml Glass Beakers</td><td>Fisherbrand</td><td>KFS14005250</td><td>Glassware</td></tr><tr><td>Glass Media Bottles With Cap</td><td>Fisherbrand</td><td>KFS14395250</td><td>Glassware (8 units)</td></tr><tr><td>50 ml Corex Tube&amp;nbsp;</td><td>Corning</td><td>8422-A</td><td>(1 unit)</td></tr><tr><td>15 ml Polystyrene Centrifuge Tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50 ml Polystyrene Centrifuge Tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>10 ml Serological Pipet</td><td>Corning</td><td>11415038</td><td></td></tr><tr><td>Cell Strainer 100 &amp;mu;m Nylon</td><td>Corning</td><td>431752</td><td></td></tr><tr><td>Absorbant Liner</td><td>Scienceware</td><td>1199918</td><td></td></tr><tr><td>500&amp;nbsp;ml Bottles Top Filter</td><td>Corning</td><td></td><td>Pore: 0.22&amp;nbsp;&amp;micro;m / medium and HBSS preparation</td></tr><tr><td>2 ml Criogenic Vials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>Freezing Container, Nalgene Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562-1EA</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Pharmacia Fine Chemicals</td><td>P3 model</td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>Model 127</td><td></td></tr><tr><td>Vacuum Pump</td><td>ABM</td><td>4EKFS6CX-4</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisherbrand</td><td>EC231-598-3</td><td>Saline solution 0.9%</td></tr><tr><td>Hank&amp;rsquo;s Buffered Salt Solution (Hbss)</td><td>Sigma-Aldrich</td><td>H2387</td><td>Quantity: 9.25 (one vial) for 1 L of digestion solution</td></tr><tr><td>Hydroxypiperazineethansulphonic Acid (Hepes)</td><td>&lt;a name=&quot;RANGE!B29&quot;&gt;Life Technologies&lt;/a&gt;</td><td>15630-080</td><td>25 ml (1 M) for 1 L of digestion solution</td></tr><tr><td>Trypsin Type I</td><td>Sigma-Aldrich</td><td>T8003</td><td>9,888 U</td></tr><tr><td>Deoxyribonuclease Type Iv</td><td>Roche</td><td>10-104-159-001</td><td>402,000 U</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td>100 mM</td></tr><tr><td>Magnesium Sulfate</td><td>Baker</td><td>2500-01</td><td>800 mM</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium High Glucose (Dmem)</td><td>Life Technologies</td><td>10564-045</td><td></td></tr><tr><td>Penicillin/Streptomycin Sulphate</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644</td><td>Density centrifugation media gradient. Volume: 36 ml</td></tr><tr><td>Isopropanol</td><td>Acros</td><td>42383-0010</td><td>50 ml</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Automacs Magnetic Separator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>Model 003</td><td></td></tr><tr><td>Automacs&amp;nbsp;Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-021-101</td><td></td></tr><tr><td>Automacs Running Buffer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-221</td><td>http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1</td></tr><tr><td>Automacs Rinsing Solution&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx</td></tr><tr><td>Anti-Human Hla Abc Purified Clone W6/32</td><td>Affymetrix eBioscience</td><td>14-9983-82</td><td>anti-mouse antibody</td></tr><tr><td>Anti Mouse Igg Microbeads</td><td>Miltenyi Biotec</td><td>130048401</td><td></td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 6 Well With Lid</td><td>Corning</td><td>3335</td><td>Cell Bind surface</td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 24 Well With Lid</td><td>Corning</td><td>3337</td><td>Cell Bind surface</td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 96 Well With Lid</td><td>Corning</td><td>3300</td><td>Cell Bind surface</td></tr><tr><td>Modular Incubator Chamber&amp;nbsp;</td><td>Billups-Rothenberg</td><td>MIC-101</td><td>A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions</td></tr><tr><td>Single Flow Meter</td><td>Billups-Rothenberg</td><td>SFM3001</td><td></td></tr><tr><td>50 mm In-Line Filter</td><td>Whatman</td><td>6721-5010</td><td>PTFE, pore: 1.0 &amp;micro;m</td></tr><tr><td>Gas Regulator</td><td>Pro Star</td><td>PRS301233</td><td>A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions</td></tr><tr><td>Gas Hose Class Vi Clear 5/16&amp;nbsp;</td><td>Parker</td><td>100-05070102</td><td>3 pieces with ~ 0.5 m</td></tr><tr><td>17 mm Adjustable Gas Hose Clamp</td><td>Tiewraps</td><td>THCSS-16</td><td></td></tr><tr><td>Normoxia Gas Cylinder&amp;nbsp;</td><td>Praxair</td><td>NI CDOXR1U-K</td><td>Size K (3&lt;sup&gt;rd&lt;/sup&gt; trimester&#039;s composition: 5% CO&lt;sub&gt;2&lt;/sub&gt;, 8% O&lt;sub&gt;2&lt;/sub&gt;, Bal. N&lt;sub&gt;2&lt;/sub&gt;)</td></tr><tr><td>Normoxia Gas Cylinder&amp;nbsp;</td><td>Praxair</td><td>NI CDOXR1U-K</td><td>Size K (3&lt;sup&gt;rd&lt;/sup&gt; trimester&#039;s composition: 5% CO&lt;sub&gt;2&lt;/sub&gt;, 0.5% O&lt;sub&gt;2&lt;/sub&gt;, Bal. N&lt;sub&gt;2&lt;/sub&gt;)</td></tr><tr><td>Oxygen Microelectrode Mi-730</td><td>Microelectrodes INC</td><td>84477</td><td></td></tr><tr><td>Oxygen Adapter</td><td>Microelectrodes INC</td><td>3572</td><td></td></tr><tr><td>ROS Detection Reagent: CM-H2DCFDA&amp;nbsp;</td><td>Invitrogen</td><td>C-400</td><td></td></tr><tr><td>&amp;beta;-hCG ELISA kit&amp;nbsp;</td><td>DRG internatinal</td><td>EIA-4115</td><td></td></tr><tr><td>Anti-Vimentin purified antibody</td><td>eBioscience</td><td>14-9897</td><td>Host: mouse</td></tr><tr><td>Anti-Cytokeratin 7 (FITC) antibody&amp;nbsp;</td><td>Abcam</td><td>ab119697</td><td>Host: mouse</td></tr><tr><td>Alexa Fluor 488 Goat Anti-mousse IgG H&amp;amp;L antibody</td><td>Life Technologies</td><td>A-11029</td><td></td></tr></tbody></table>

human primary trophoblast cell culture model to study the protective effects of melatonin against hypoxia/reoxygenation-induced disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

分离和绒毛细胞滋养层细胞的免疫纯化从经阴道分娩获得正常足月胎盘产生1×10 8个活细胞。胎盘称重350克，为19厘米，直径4厘米高与圆盘状和透明膜。没有检测到子叶畸形。脐带有旁本地化和56厘米的长度。的纯度通过流式细胞术使用波形蛋白和细胞角蛋白7的标记。细胞的98％以上是阴性的波形和阳性细胞角蛋白7，确认从免疫纯化获得的绒毛滋养细胞的纯度。绒?...

Watch this Scientific Journal Video about Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption at JoVE.com

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

人胎盘滋养层细胞培养模型，研究褪黑素对缺氧的保护作用/复氧引起的中断

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

human-primary-trophoblast-cell-culture-model-to-study-protective

Research

JoVE Journal

Developmental Biology

15.8K 次观看.  INRS-Institut Armand-Frappier. This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

视频: Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

生物工程

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

发育生物学

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

评估高氧对人新生儿气道上皮细胞影响的转化 3D 细胞培养模型

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

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 ...

生物学

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

人体滋养层模型研究母体肥胖炎症对胎盘自噬的调节作用

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

医学

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

人类心脏肌细胞模型中体温过低预科后心肌保护体外评估

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...