这项工作是由来自研究基金会 - 弗兰德（FWO G.0856.13N）和鲁汶研究理事会（OT / 13/113）资助。 KDC由比利时FWO资助。 AH被OT /一百十三分之十三资助。我们要感谢的鲁汶（http://gbiomed.kuleuven.be/english/corefacilities/microscopy/cic/cic.htm）的细胞成像核心设施的利用共聚焦显微镜。

AlexaFluor 这项研究的所有动物实验由鲁汶（比利时）（项目P174 / 2013）的伦理审查委员会的批准。小鼠在标准条件下收容，与食物小丸随意访问和自来水，并保持在受控条件（23±1.5℃，相对湿度40 - 60％，12/12光/暗循环）下。 1.小鼠<ol><li>使用市售的小鼠品系（ 如 C57BL / 6，女8 - 19周龄）。通常情况下，4 - 5只将产生的细胞用于进一步的实验的一个适当的量。 </li><li>注入皮下完好的小鼠用100微升17β雌二醇（E2）溶液（100毫微克/ 100μL）连续3 d后再隔离为了规范动物的发情周期的阶段，并诱发子宫内膜增生细胞。需要用于启动协议之前检查小鼠的周期相是可以避免的BE3 D&#39;雌二醇病因治疗。 </li></ol> 2.准备<ol><li>使100毫微克/ 100微升的E2在花生油中的溶液。对五只小鼠（如在1.2中描述）的注射剂，需要1.5毫升的量。 <ol><li>溶解1毫克的E2在1mL乙醇至1毫克/毫升的储备溶液。 </li><li>在花生油1000:这个稀释原液1。 </li></ol></li><li>使500 Hank氏平衡盐溶液（HBBS）1×100单位/ mL青霉素和100微克/毫升链霉素补充的毫升（进一步称为HBSS +）。 </li><li>使过滤小鼠子宫内膜基质细胞的500毫升（卓制）培养基培养卓制:Dulbecco改进的Eagle培养基/营养混合物F12（DMEM / F12）用酚红，L-谷氨酰胺和4-（2-羟乙基）哌嗪-1-乙磺酸酸，N - （2-羟乙基）哌嗪-N&#39; -含有10％FBS，0.5微克/毫升两性霉素B，和100微克/毫升庆大霉素（2-乙磺酸）（HEPES）（福rther称为卓制介质）。 </li><li>使500毫升的过滤2％卓制培养基培养卓制蜕膜化过程中:DMEM / F12用酚红，L-谷氨酰胺和含有2％FBS，0.5微克/毫升两性霉素B的HEPES，和100微克/毫升庆大霉素。 </li><li>制备过滤小鼠子宫内膜上皮细胞的500毫升（MEEC）培养基:含有10％FBS，0.5微克/毫升两性霉素B，100微克/毫升庆大霉素，25％MCDB-105培养基中，5微克/毫升胰岛素的DMEM（进一步称为作为MEEC介质）。 </li><li>制备用于卓制培养涂覆聚L-赖氨酸（PLL）的盖玻片。 <ol><li>溶解25mg的锁相环在250毫升的蒸馏水。过滤用0.22微米过滤器的溶液。 </li><li>在一个12孔板添加无菌盖玻片。添加在盖玻片溶解PLL的300微升。 </li><li>温育后的30分钟，除去该溶液中。所述板可以储存在4℃直至进一步使用（高达1 - 2月）。 </li></ol></li><li>准备一个10毫克/毫升原液Ø˚F胶原酶在-20°C IA和存储等分的300微升。使用前过滤。 </li></ol> 3.筹备24小时之前分离所需要的<ol><li>准备胶原蛋白涂盖玻片MEEC文化。 <ol><li>制备在蒸馏水中的0.02M乙酸溶液（每盖玻片1mL）中。 </li><li>添加150μL的在12ml胶原的0.02M乙酸，得到50微克/毫升的最终浓度。 </li><li>在一个12孔板添加无菌盖玻片。 </li><li>加入1毫升的胶原溶液（见3.1.2）。 </li><li>在37℃孵育O / N（最少12小时）。 在隔离: </li><li>通过抽吸除去落盖玻片胶原溶液，让它在无菌环境（在层流柜）风干。 </li><li>用磷酸盐缓冲盐水（PBS）洗涤盖玻片两次。 </li></ol></li><li>通过在含有10毫升15毫升规范管加入0.25克胰酶制备2.5％胰酶溶液HBSS +。摇动（200转），在37℃的溶液1小时，以增加溶解度。保存在4℃。 </li></ol> 4.分离（MEEC）鼠标子宫内膜上皮细胞的培养和小鼠子宫内膜基质细胞（MESC） <ol><li>加入25毫克的胰蛋白酶在含2.5％胰酶溶液（见3.2）用含有0.25％胰蛋白酶的最终溶液（还称为卓制消化混合物）来结束一个15毫升管中。 </li><li> 子宫的分离 <ol><li>检查动物的周期阶段有阴道涂片检查。 <ol><li>抑制动物，并用50微升PBS中的3轻轻冲洗阴道 - 5倍。 </li><li>收集在载玻片上最后的冲洗。 </li><li>检查使用10X或20X的目标明场显微镜下的材料。 </li><li>只使用在发情期的小鼠。这个阶段的特点是在阴道灌洗角化上皮细胞的存在（ 图1 </strong&gt;的）。 </li></ol></li><li>安乐死使用适当的方法，如颈椎脱位或CO 2诱导麻醉动物。 </li><li>喷用70％乙醇的屠体，以产生一个无菌环境。 </li><li>使用无菌手术工具，打开腹部和推动肠一边想象这两个子宫角。 </li><li>抢角子宫的输卵管在最远端。从输卵管解剖子宫角和去除脂肪和结缔组织。剪下喇叭在子宫体上方的远端，并放置在35毫米培养皿3毫升HBSS +的。 <ol><li>直到所有的子宫角收集重复此步骤进行其他喇叭和其他动物。 </li></ol></li><li>清洁子宫角（在HBSS +）进一步下一个立体镜，并删除所有剩余的脂肪和结缔组织。 </li><li>切开子宫角开放纵向暴露子宫腔和更换日在一个新的培养皿新鲜HBSS + E号角。 </li><li>传输所有子宫角含有胰酶和胰蛋白酶的15毫升管。 </li><li>在4℃下在轨道摇床（50rpm下）水平地孵育60分钟。 </li><li>水平孵育在23℃（RT）45分钟，不摇动。 </li><li>水平孵育在37℃下（水浴）15分钟，不摇动。 </li><li>通过在2.7毫升0.05％胰蛋白酶乙二胺四乙酸（EDTA）的溶液中溶解1毫克/毫升胶原酶库存300微升同时使卓制消化混合物。 注:从现在起，在无菌环境中的层流柜下进行进一步的隔离措施。 </li></ol></li><li> 鼠标子宫内膜上皮细胞（MEEC）文化 <ol><li>孵育2小时后，小心地倒出上清液溶液。 </li><li>转移子宫到含有冷MEEC介质培养皿并孵育5分钟，以灭活胰蛋白酶活动。 </li><li>转移子宫含有3毫升冷HBSS +的15毫升管中。 </li><li>涡旋10秒以释放上皮片。 </li><li>在冲洗干净培养皿3毫升HBSS +子宫。 </li><li>重复步骤4.3.2 - 4.3.4以获得总的含有上皮片3的悬浮液。 </li><li>转移子宫到卓制消化混合，并在37℃下孵育30分钟，同时振摇（200转）（转到步骤4.4卓制的进一步隔离）。 </li><li>通过以除去组织碎片轻轻吹打三种细胞悬浮液上的100微米的尼龙筛恢复上皮片。 </li><li>离心在500 xg离心所收集的细胞悬浮液5分钟。 </li><li>重悬在12毫升MEEC介质的颗粒拌匀。 </li><li>沉降5分钟的溶液中，以使用重力沉降分离剩余卓制。 </li><li>用5毫升吸管小心地取出上部2毫升。 </li><li>离心机细胞悬索桥Ñ在500×g离心5分钟。 </li><li>悬浮在MEEC介质中轻轻吹打起来。 </li><li>板在进一步的实验（ 图2a）所需的密度胶原涂层盖玻片细胞。 </li><li>孵育所述细胞在37℃在一个5％CO 2培养箱中培养最少12小时。 注:对于免疫染色或功能实验，如荧光钙成像，建议直接板上的细胞上的盖玻片，而RNA或蛋白质分离期望时，建议在一个6孔板接种。小鼠子宫内膜间质细胞的分离是只有一个消化后分成两轮作为细胞的纯度可以是不足的。将子宫一个最佳的细胞恢复和纯度消化两次。在这两轮的技术措施是相同的。从两轮收集的细胞可保持用于进一步的实验中，由研究人员根据需要。 </li></ol></li><li> 鼠标子宫内膜海峡OMAL细胞（MESC）文化: 第一轮<ol><li>第一轮消化的开始之前，准备在2.7毫升0.05％胰蛋白酶EDTA溶液溶解1毫克/毫升胶原酶股票300μL第二消化组合。这样的搭配需要在步骤4.4.8。 </li><li>用冷的（4℃）的HBSS +和3×15用3mL卓制介质毫升管（管X1，X2和X3）准备三个小培养皿中。 </li><li>温育30分钟后，振摇10秒含子宫胰蛋白酶轻轻溶液。卓制细胞将从子宫组织分离并将保持在胰蛋白酶溶液中。 </li><li>转移子宫到含有3毫升冷（4℃）的HBSS +第一培养皿和冲洗干净。 </li><li>加入3毫升卓制介质与原本含有子宫角（管在步骤4.4.3）管抑制胰蛋白酶活性。 </li><li>从培养皿用冷（4℃）转移子宫HBSS +含3毫升卓制介质的管X1并轻轻摇晃10秒。 </li&gt; <li>重复步骤4.4.4（转移至培养皿用冷（4℃）的HBSS +）和4.4.6（转移至管X2和X3）的两倍。 注:最后，该协议将导致4细胞悬浮液:含有胰蛋白酶溶液并用含有卓制卓制介质3管（管X1，X2和X3）一个管。 </li><li>在新的卓制消化转移子宫，如在步骤4.4.1中描述，并在37℃下孵育30分钟，垂直。 </li><li>通过一个40微米尼龙网传递的四个细胞悬液收集基质细胞。冲洗网与另外5毫升卓制。 </li><li>离心在500 xg离心细胞悬浮液7分钟。 </li><li>悬浮于PLL的涂覆的盖玻片为具有所需密度的进一步实验中卓制和板沉淀。 </li><li>孵育至少4 -在5％CO 2培养箱6小时或O / N在37℃。 </li></ol></li><li> 小鼠子宫内膜基质细胞（MESC）文化: 第二轮 &lt;OL&gt; <li>用冷的（4℃）的HBSS +和3×15用3mL卓制介质毫升管（管Y1，Y2和Y3）准备三个小培养皿中。 </li><li>重复步骤4.4.3 - 4.4.7。 </li><li>与子宫到一个40微米尼龙网一起传送的最后一个细胞悬浮液。 </li><li>通过使管Y1，Y2和Y3的内容通过尼龙网收集基质细胞。冲洗网与另外5毫升卓制。 </li><li>离心在500 xg离心细胞悬浮液7分钟。 </li><li>悬浮于PLL的涂覆的盖玻片进一步的实验（ 图2b）中卓制介质和板沉淀。 </li><li>孵育至少4 - 37℃6小时，用5％ 的 CO 2。 注:对于免疫染色或功能实验，如荧光钙成像，建议直接制版盖玻片上的细胞，而在一个6孔板接种RNA或蛋白质分离期望时，建议。 </li></ol></li></ol> 5.波形蛋白/角蛋白双染纯MEEC和卓制文化的验证<ol><li>种子盖玻片上的细胞。 </li><li>洗3×5分钟用含有的钙和镁的PBS在轨道摇床（50rpm下）。 </li><li>用4％多聚甲醛（PFA）（ 小心！）的细胞10分钟，而不是在摇床上。 </li><li>洗3次，用PBS而不会对摇床（50rpm下）钙和镁。 </li><li>透用0.2％的Triton X-100将细胞在振荡器（50rpm）进行10分钟。 </li><li>在摇床上洗涤3次，用PBS。 </li><li>用5％山羊血清（GS）在PBS中2小时振荡器上块（50rpm）进行</li><li>在4℃在振荡器上（50rpm）进行孵育单克隆兔抗人波形蛋白的细胞（1:500）和单克隆小鼠抗人pancytokeratin（1000 1）。抗体稀释在PBS中的0.5％葡萄糖。 </li><li>洗3次，用PBS振荡器上（50rpm下）。 </li><li>孵育细胞用第二抗体（1:1000于0.5％GS）的Alexa Fluor 488缀合的抗兔IgG和Alexa Fluor 488- conju在摇床上门控的抗小鼠IgG。 </li><li>在摇床上洗涤3次，用PBS。 </li><li>滑山含DAPI和干燥的O / N水性封固剂。 </li><li>密封用指甲油的幻灯片，并保持在4℃，为进一步利用（ 图2A，2B）。 </li></ol> 6. 在体外蜕膜在共同培养体系注:四到五只动物中需要一个24孔板以建立共培养系统。用在无菌环境下协议继续，优选在层流柜中。 <ol><li>期间在额外的24孔平板的上皮细胞分离（如在4.3节中描述）的离心和/或温育步骤制备的细胞培养插入物。 <ol><li>添加200 - 卓制介质的400微升到在24孔板的孔的所需量。 </li><li>放置插入使用无菌镊子预充式24井。 </li></ol></li><li>悬浮颗粒MEEC在通过插入MEEC中，除（步4.3.14）（工作容积:150 - 每个刀片300μL）。培养在5％CO 2培养箱将细胞在37℃。 </li><li>第二轮的基质细胞分离的（步骤4.5.6）后的种子在另一个24孔板的基质细胞（与细胞培养插入兼容）。用每孔400μL的细胞悬浮液的工作容积。 </li><li>允许基质细胞为至少4附上- 6小时，在37℃在一个5％CO 2培养箱。 </li><li>转移覆盖有上皮细胞从最初的24孔板的细胞培养插入到由基质细胞所覆盖的第2个24孔板中。使用消毒镊子或者只在他们的脚下挂着触摸刀片。 </li><li>培养在5％CO 2培养箱一段3的细胞，在37℃ - 5 d，直到上皮细胞形成单层和基质细胞达到80 - 90％的汇合。此外，使用跨膜电阻（TEER）通过使用电压/欧姆上皮计如由Grant 等人描述并监控上皮单层。 7。 800-1000 /孔值足以考虑单层上皮融合。 </li><li>用玻璃吸管或细枪头3天 - 如果有必要，则更换介质每2。开始更换基质细胞（在底部接种）的培养基中，在插入件更换培养基之前。建议在37℃下用预热的细胞培养基。 </li><li>准备蜕膜中开始实验前在体外蜕膜化诱导。使用每个基质细胞井400微升的工作容积。 <ol><li>准备在-20°C以下的股票解决方案和存储等分。 <ol><li>制备在乙醇250μM甲羟孕酮-17-乙酸酯（MPA）。 </li><li>制备的100mM储备溶液-8- Bromoadenosine 3&#39;，5&#39;-环磷酸腺苷在超纯水中。 </li></ol></li><li>使含有1μM的MPA和0.5mM的cAMP在含有2％FBS的卓制介质蜕膜介质。 </li></ol></li><li>轻柔地从井介质，并添加蜕膜中到间质细胞。如果需要控制条件，使用含有2％FBS的MESC网上平台。 </li><li>从细胞培养插入物除去介质并加入MEEC介质300微升到细胞上。 </li><li>孵育5天的细胞在培养箱中在37℃在5％CO 2。 </li><li>第五天后，评估在通过RT-qPCR的基质细胞蜕膜的程度（见下文）。 </li><li>验证使用基于刃天青生存力试剂上皮细胞的生存力。 <ol><li>制备活力试剂在MEEC介质1:10溶液。 </li><li>转移的细胞培养插入到一个新的24孔板中。 </li><li>添加150 - 活力试剂的300微升 - MEEC溶液到细胞上。 </li><li>在5％CO 2在孵育在37℃下至少4 - 5小时或过夜，并通过观察色移（蓝色至紫色/粉红色）评估细胞生存力。 注意:蜕膜也可以使用特定的催乳素小鼠ELISA来评估，由研究人员为优选的。 </li></ol></li></ol>蜕膜的7.Validation定量RT-PCR 注:对于通过qRT-PCR的蜕膜的验证，所以建议以执行，以便接收单元的足够量的RNA分析中重复所有的试验条件。 如。德克拉克等人先前所述进行定量RT-PCR。 8。 简单来说: <ol><li>隔离用市售RNA提取试剂盒提取总RNA。 <ol><li>评估使用根据制造商的协议的商业方法RNA的数量和质量，分别。 </li></ol></li><li>合成cDNA，根据制造商的协议从总RNA的1微克开始。 </li><li>使用根据制造商的协议的商业试剂盒来进行定量RT-PCR反应。 <ol><li>一式三份运行的所有样本。 </li><li>使所需看家基因和催乳素（prl8a2）利用商用的TaqMan主混合物和特异性TaqMan探针来进行该反应。 </li></ol></li></ol>

<ol>
	<li>Cummings, A. M., Yochim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+uterus+in+preparation+for+gestation:+a+model+for+the+action+of+progesterone.">Differentiation of the uterus in preparation for gestation: a model for the action of progesterone.</a> J Theor Biol. 106 (3), 353-374 (1984).</li><li>Jabbour, H. N., Kelly, R. W., Fraser, H. M., Critchley, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+regulation+of+menstruation.">Endocrine regulation of menstruation.</a> Endocr Rev. 27 (1), 17-46 (2006).</li><li>Wang, X., Matsumoto, H., Zhao, X., Das, S. K., Paria, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signals+direct+the+formation+of+tight+junctional+permeability+barrier+in+the+decidualizing+stroma+during+embryo+implantation.">Embryonic signals direct the formation of tight junctional permeability barrier in the decidualizing stroma during embryo implantation.</a> J Cell Sci. 117 (Pt 1), 53-62 (2004).</li><li>Zygmunt, M., Herr, F., Munstedt, K., Lang, U., Liang, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+vasculogenesis+in+pregnancy.">Angiogenesis and vasculogenesis in pregnancy.</a> Eur J Obstet Gynecol Reprod Biol. 110 Suppl 1, S10-S18 (2003).</li><li>Power, M. L., Schulkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The evolution of the human placenta. , (2012).</li><li>Finn, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initiation+of+the+decidual+cell+reaction+in+the+uterus+of+the+aged+mouse.">The initiation of the decidual cell reaction in the uterus of the aged mouse.</a> J Reprod Fertil. 11 (3), 423-428 (1966).</li><li>Grant, K. S., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mouse+uterine+stromal+cells+on+epithelial+cell+transepithelial+resistance+(TER)+and+TNFalpha+and+TGFbeta+release+in+culture.">Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture.</a> Biol Reprod. 69 (3), 1091-1098 (2003).</li><li>De Clercq, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+transient+receptor+potential+channels+in+human+endometrial+stromal+cells+during+the+luteal+phase+of+the+menstrual+cycle.">Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.</a> Hum Reprod. 30 (6), 1421-1436 (2015).</li><li>Pieper, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vimentin+expression+in+cultured+epithelial+cells.">Regulation of vimentin expression in cultured epithelial cells.</a> Eur J Biochem. 210 (2), 509-519 (1992).</li><li>Greaves, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+endometriosis+mimics+human+phenotype+and+reveals+insights+into+the+inflammatory+contribution+of+shed+endometrium.">A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.</a> Am J Pathol. 184 (7), 1930-1939 (2014).</li><li>Pierro, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+interactions+modulate+estrogen+responsiveness+in+normal+human+endometrium.">Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium.</a> Biol Reprod. 64 (3), 831-838 (2001).</li><li>Kim, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone-dependent+release+of+transforming+growth+factor-beta1+from+epithelial+cells+enhances+the+endometrial+decidualization+by+turning+on+the+Smad+signalling+in+stromal+cells.">Progesterone-dependent release of transforming growth factor-beta1 from epithelial cells enhances the endometrial decidualization by turning on the Smad signalling in stromal cells.</a> Mol Hum Reprod. 11 (11), 801-808 (2005).</li><li>Chen, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coculturing+human+endometrial+epithelial+cells+and+stromal+fibroblasts+alters+cell-specific+gene+expression+and+cytokine+production.">Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production.</a> Fertil Steril. 100 (4), 1132-1143 (2013).</li><li>Renaud, J., Martinoli, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Insert+Co-culture+System+of+Two+Cellular+Types+in+the+Absence+of+Cell-Cell+Contact.">Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact.</a> J Vis Exp. (113), (2016).</li><li>Arnold, J. T., Kaufman, D. G., Seppala, M., Lessey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+stromal+cells+regulate+epithelial+cell+growth+in+vitro:+a+new+co-culture+model.">Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model.</a> Hum Reprod. 16 (5), 836-845 (2001).</li><li>Park, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+well-defined+in+vitro+three-dimensional+culture+of+human+endometrium+and+its+applicability+to+endometrial+cancer+invasion.">A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.</a> Cancer Lett. 195 (2), 185-192 (2003).</li></ol>

作者什么都没有透露。

蜕膜是子宫内膜间质细胞的孕酮依赖性分化成圆形分泌蜕膜细胞。在人类，该方法在月经周期的黄体期自发发生，并且在围绕血管细胞以形成predecidua基质细胞被启动。然而，在啮齿动物中，胚泡的存在是必要的，以诱导蜕膜。该蜕膜仅与子宫内膜上皮细胞接触后发生的事实意味着，重要的因素是由上皮细胞分泌诱导蜕膜在下面的基质。虽然在蜕膜过程开始这种差异应该承认，即人的蜕膜变得在胚胎着床更健壮的事实表明，类似的机制可以参与。 体外细胞培养物经常用于研究蜕膜的过程中特定分子的效果。尽管如此，可收集的可用性和人体组织的量通常是有限的，并伴随着变化， 例如，周期相，怀孕次数和妇科疾病，如子宫内膜异位症或子宫腺肌病存在。许多这些问题都可以通过使用鼠组织容易触及和独立周期阶段来预防。用小鼠组织的另一个强大的优势是使用转基因老鼠的机会，并有可能建立了大量的实验。此刻，许多不同的隔离协议已经在文献中，在动物的年龄高变异性，并在使用的时刻在发情期的期间中所述。 这项研究提出了基质和上皮细胞，其中的优势是由现有的隔离每日注射雌激素所取的标准化发情周期的初级内膜共培养物的新方法。此外，雌激素为known诱导增殖的上皮和间质细胞，这进一步增加每隔离的产率。本文中所描述的隔离协议基于Grant 等。 7，但是，被包括进一步的优化步骤，以提高产率，纯度，以及不同的细胞培养物的生存力。首先，HBSS总是补充有抗生素，以避免原代培养的污染。期间MEEC的隔离，一个温度适应已完成（在37℃下15分钟），以增加所述第一消化过程中的上皮细胞的收获。接着，一个附加的步骤中包括通过加入培养基含有FBS的以抑制其活性的胰蛋白酶消化后回火酶活性。此外，MEECs是通过一个过滤器的网孔比是通常描述（100微米），因为它们常常是在集群和细胞片较大传递。此外，通过基质细胞污染减少了执行一个附加itional步骤，其中MEECs和MESCs是基于重力沉降分离出来。最后，MEECs接种在胶原包被的盖玻片，以增加细胞对盖玻片附件，因为很明显，附着到未涂覆或PLL涂覆的玻璃盖玻片是不够的。期间MESCs的隔离，胶原酶（1毫克/毫升）进行了补充，以改善消化。此外，该消化步骤重复进行，以避免残留MEECs的污染。所有这些额外的步骤导致同质细胞群的纯净和可行的文化。 细胞群的纯度用免疫染色和定量RT-PCR，使用波形蛋白作为基质标记物和细胞角蛋白作为上皮标记物进行了评价。显然，在MEEC和卓制观察波形蛋白和细胞角蛋白的相对表达模式。然而，可以注意到，波形蛋白和细胞角蛋白的相对mRNA表达是在MEEC培养物相似。 Simil芳结果由其他研究小组发表的，其中上皮细胞中培养的获取波形蛋白表达9。更重要的是在波形蛋白和如在不同的细胞群的免疫组织学染色观察细胞角蛋白之间的蛋白表达的差异。双重染色显示，EECS阳性细胞角蛋白和ESC呈阳性波形。在免疫染色中使用这些标记物的是一个既定的方法和标准地用于指示细胞类型10。 虽然大量的研究已在卓制的蜕膜进行，所以该上皮细胞在此模型中的存在可能会改变蜕膜的结果仍然是可能的。首先，已经表明，如果MEEC生长到单层中卓制条件培养基的存在下或在共培养设置，该单层具有改进的上皮细胞的跨上皮电阻（TEER），由卓制7分泌的因素造成。此外，Pierro 等。 11提供的证据表明，上皮细胞增殖，通过17β雌二醇的影响，可能会被从所述基质细胞分泌的因子介导的，并且可替代地，上皮细胞可以释放该调制基质蜕膜12,13的因素。总体而言，很明显，上皮 - 间质共培养环境提供了更多的生理状况，它允许旁分泌相互作用和通信。被描述采用一个插入共培养系统中培养两种不同的细胞类型的方法更早14,15和被改编为使用鼠原代细胞。由检测催乳素mRNA水平作为读出对蜕膜的，所描述的技术具有可和允许非常可重复的读出为蜕膜Ş从而蜕膜化过程中的上皮 - 间质关系的研究。从MEEC介导的旁分泌信号可能改变蜕膜化程度的间质细胞。此外，该模型允许上皮侧的特定调制而不直接影响基质细胞。因此，MEEC和卓制的此共培养提供了评估激素，细胞因子等对上皮细胞上的基质蜕膜一个读出的效果的理想工具。此共培养体系的另一个优点是，它允许研究人员研究的特定蛋白在蜕膜过程中任一上皮或间质隔室通过使用从转基因动物分离的细胞的参与。此外，这种技术将是非常有益的，调查胚泡粘合性评价通过上皮细胞的胚泡的存在下分泌的旁分泌因子是否足以诱导底层基质的蜕膜细胞。在滋养细胞浸润的一个重要步骤相关的细胞外基质降解，这是由蛋白水解酶的作用介导的。所提出的技术也可应用于作为体外模型来研究胚泡，上皮细胞和支承基质之间的串扰。 然而，此刻知之甚少的上皮细胞可以是如腺以及管腔的起源。因此，需要的上皮细胞的遗传和分子分布的进一步表征。此外，所描述的方法是在对上皮细胞的偏振的可用知识的限制。此刻，上皮细胞的偏振没有考虑。然而，在膜蛋白表达的差异在顶和基膜进行说明。上皮细胞层的不适当偏振可能对epitheli之间的旁分泌相互作用的冲击人与基质细胞。然而，方法获得极化上皮细胞中已被描述，并且可以包括在所描述的技术15,16。 总之，所描述的协议提出的技术成功建立小鼠子宫内膜上皮细胞和基质细胞原代细胞培养。这种技术在纯细胞培养物的结果通过qRT-PCR和波形蛋白和细胞角蛋白的免疫染色证实。最终来说，上皮细胞和基质共培养引进，允许通过任何MEEC和/或卓制在蜕膜过程介导的旁分泌信号的调查。

人子宫内膜子宫内膜和经历击穿和修理的每月的周期作为可能怀孕的制剂。它由上皮细胞衬子宫腔和根据卵巢激素的雌激素和孕激素的波动厚度而变化下面的基质的单层的。在黄体期，在排卵后孕酮浪涌会诱发子宫内膜间质细胞分化成较大，圆形蜕膜细胞，一个称为蜕膜1,2的过程。在人的，这种现象会出现自发形成predecidua，而是成为在胚胎着床更加明显。蜕膜的功能性后果是，子宫暂时变成接受胚胎着床。这个间隔被标记为"植入窗口"。在胚胎着床的初期，蜕膜ê包围植入胚胎ndometrial基质细胞将提供一个屏障，以防止有害物质的通道的胚胎3。在怀孕期间，这将蜕膜对发育中的胎儿提供营养胎盘已经发生之前，稍后会形成胎盘4的母体部分。 道德和实际的考虑限制人类研究的设计，调查蜕膜的进程，并促使用动物模型。作为hemochorial胎组的成员，啮齿动物的子宫内膜也将经历前胎5蜕膜。在啮齿动物，然而，蜕膜处理的执行依赖于在子宫腔囊胚的存在，这表明一个额外的刺激是必要触发蜕膜响应6。这意味着日之间错综复杂的通信Ë具有与胚泡直接接触子宫内膜上皮细胞，和底层基质细胞。然而，蜕膜可人为使用刺激机械划伤或油中激素催芽子宫灌注诱导。虽然在体内研究采用人工蜕膜化模型使我们能够改善我们的见解蜕膜中的复杂的信号处理，机械深入的研究， 例如，上皮细胞和间质细胞之间的通信不可缺少的调查，是有限的。因此， 在体外蜕膜研究可以用于操纵重要途径和研究基本过程。为此，主子宫内膜间质细胞培养物可以从人或啮齿动物子宫内膜建立。用人鼠类同意使用转基因动物的研究感兴趣的特定蛋白在蜕膜化过程中的作用。然而，不成熟，prepuberTY小鼠经常使用，以避免在发情周期的并发症，而在其他情况下，子宫从动情周期，这导致在培养一个异质的所有阶段收集。此外，蜕膜的过程已经在不同的协议， 例如，研究了通过（i）补充激素（雌激素，孕激素或环磷酸腺苷（cAMP）的），以在培养介质中，或由（ⅱ）在蜕膜细胞的分离从小鼠（伪）怀孕，其需求插头检查和输精管结扎男性额外需要4.5日子。这些限制展示出一种标准化方法的必要性在体外蜕膜化学习。在这项研究中，生理模型来研究体外蜕膜从成年起，无（假）孕鼠将提交。在该方法中，每日注射17β雌二醇（E2）的会诱发子宫内膜细胞的增殖，导致均匀和p增加的产量URE细胞群。此外，使用的共培养系统的允许上皮 - 间质串扰和操纵不同层次蜕膜的过程的能力的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ThinCert Tissue culture insert for 24 well plates; 1 &#181;m pore size; transparant</td>
			<td>Greiner Bio-one</td>
			<td>662610</td>
			<td>Inserts are also provided for other multiwell plates and/or other pore sizes. Other suppliers: Merck Millipore. 
			https://shop.gbo.com/en/row/articles/catalogue/article/0110_0110_0090_0030/13408/&#160;</td>
		</tr>
		<tr>
			<td>Nunc Cell culture treated 24 well plates</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td> http://www.thermofisher.com/order/catalog/product/142475&#160; </td>
		</tr>
		<tr>
			<td>8-Bromoadenosine 3&#8242;,5&#8242;-cyclic monophosphate sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>B7880</td>
			<td>Prepare a stock solution of 100 mM in Milli Q water. Store aliquots at -20 &#176;C. http://www.sigmaaldrich.com/catalog/product/sigma/b7880?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Medroxyprogesterone 17-acetate</td>
			<td>Sigma Aldrich</td>
			<td>M1629</td>
			<td>Prepare a stock solution of 250 &#181;M in ethanol. Store aliquots at -20 &#176;C. http://www.sigmaaldrich.com/catalog/product/sigma/m1629?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Arachis oil</td>
			<td>Supermarket</td>
			<td></td>
			<td>Standard cooking arachis oil that can be found in every supermarket is used </td>
		</tr>
		<tr>
			<td>PrestoBlue cell viability reagent</td>
			<td>Thermo Scientific</td>
			<td>A13261</td>
			<td> https://www.thermofisher.com/order/catalog/product/A13261?ICID=cvc-prestoblue-c1t1&#160; </td>
		</tr>
		<tr>
			<td>HBSS 1x, no calcium, no magnesium, no phenol red</td>
			<td>Gibco</td>
			<td>14175-046</td>
			<td>Store in fridge as long as possible. Use cold HBSS+ during the protocol. https://www.thermofisher.com/order/catalog/product/14175046&#160; </td>
		</tr>
		<tr>
			<td>17-&#946; Estradiol</td>
			<td>Sigma Aldrich</td>
			<td>E8875</td>
			<td>Prepare a stock solution of 1mg/ml in EtOH before diluting in arachis oil (1:1000). http://www.sigmaaldrich.com/catalog/product/sigma/e8875?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Cell medium filter Stericup, 0.22 &#181;m, polyethersulfone, 500 mL, radio-sterilized</td>
			<td>Merck Millipore</td>
			<td>SCGPU05RE</td>
			<td> http://www.merckmillipore.com/BE/en/product/Stericup-GP%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-500%C2%A0mL%2C-radio-sterilized,MM_NF-SCGPU05RE&#160; </td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5,000 U/mL)</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td> https://www.thermofisher.com/order/catalog/product/15070063?ICID=search-15070-063&#160;&#160;&#160;&#160; </td>
		</tr>
		<tr>
			<td>DMEM/F12, phenol red, L-glutamine, HEPES</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td> https://www.thermofisher.com/order/catalog/product/11330057?ICID=search-11330-057&#160; </td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin</td>
			<td>Gibco</td>
			<td>10500-056</td>
			<td> https://www.thermofisher.com/order/catalog/product/10500064?ICID=search-10500-064&#160; </td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td> https://www.thermofisher.com/order/catalog/product/15290018?ICID=search-15290-018&#160; </td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/ml)</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td> https://www.thermofisher.com/order/catalog/product/15750037?ICID=search-15750-037&#160; </td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td>Sigma Aldrich</td>
			<td>D5921</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/d5921?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>MCDB-105</td>
			<td>Sigma Aldrich</td>
			<td>M6395</td>
			<td>Dilute in Milli Q water, adjust pH to 7 with NaOH and filter before use. Store in the dark. http://www.sigmaaldrich.com/catalog/product/sigma/m6395?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>I1882</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/i1882?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Coverslips, glass, 18 mm &#216;</td>
			<td>Glaswarenfabrik Karl Hecht</td>
			<td>1001/18</td>
			<td> http://www.hecht-assistent.de/187/?L=1&#38;tx_rmproducts_pi1[g]=937&#38;tx_rmproducts_pi1[p]=3504&#38;tx_rmproducts_pi1[v]=20088&#38;cHash=abd12065683fe74b00eaa5e562824d06&#160; </td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma Aldrich</td>
			<td>P2636</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/p2636?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Collagenase type IA from Clostridium histolyticum</td>
			<td>Sigma Aldrich</td>
			<td>C2674</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/c2674?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td> https://www.thermofisher.com/order/catalog/product/14190094?ICID=search-14190-094&#160; </td>
		</tr>
		<tr>
			<td>Trypsin from bovine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>T7409</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/t7409?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Warm to room temperature before use. https://www.thermofisher.com/order/catalog/product/25300054?ICID=search-25300-054&#160; </td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m mesh, disposable</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td> https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680&#160; </td>
		</tr>
		<tr>
			<td>Cell strainer, 100 &#181;m mesh, disposable</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td> https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680&#160; </td>
		</tr>
		<tr>
			<td>Millex-GP Syringe Filter Unit, 0.22&#160;&#181;m, polyethersulfone, 33&#160;mm, gamma sterilized</td>
			<td>Merck Millipore</td>
			<td>SLGP033RS</td>
			<td> https://www.merckmillipore.com/BE/en/product/Millex-GP-Syringe-Filter-Unit%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-33%C2%A0mm%2C-gamma-sterilized,MM_NF-SLGP033RS?bd=1&#160; </td>
		</tr>
		<tr>
			<td>Acetic acid (glacial) 100%</td>
			<td>Merck Millipore</td>
			<td>1.00063</td>
			<td> https://www.merckmillipore.com/BE/en/product/Acetic-acid-%28glacial%29-100%25,MDA_CHEM-100063&#160; </td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Corning&#160;</td>
			<td>354236</td>
			<td>From rat tail. https://catalog2.corning.com/LifeSciences/en-BR/Shopping/ProductDetails.aspx?categoryname=&#38;productid=354236%28Lifesciences%29#&#160; </td>
		</tr>
		<tr>
			<td>Pancreatin from porcine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>P3292</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/p3292?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>35 mm Tissue Culture Dishes, Polystyrene, Sterile</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td> https://us.vwr.com/store/catalog/product.jsp?product_id=4675630&#160; </td>
		</tr>
		<tr>
			<td>Ethanol absolute AnalaR NORMAPUR ACS, Reag. Ph. Eur. analytical reagent</td>
			<td>VWR Chemicals</td>
			<td>20821296</td>
			<td> https://uk.vwr.com/store/catalog/product.jsp?catalog_number=20821.310DP&#160; </td>
		</tr>
		<tr>
			<td>monoclonal rabbit anti-human vimentin (D21H3)&#160;</td>
			<td>Cell Signalling Tech</td>
			<td>#5741</td>
			<td>use 1/500. http://www.cellsignal.com/products/primary-antibodies/vimentin-d21h3-xp-rabbit-mab/5741 </td>
		</tr>
		<tr>
			<td>monoclonal mouse anti-human pan cytokeratin&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C2562</td>
			<td>use 1/1000. http://www.sigmaaldrich.com/catalog/product/sigma/c2562?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution, 4% in PBS</td>
			<td>Affymetrix</td>
			<td>19943</td>
			<td> http://www.affymetrix.com/catalog/130621/USB/Paraformaldehyde+Solution+4+percent+in+PBS#1_1&#160; </td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td> http://www.sigmaaldrich.com/catalog/product/SIAL/X100?lang=en&#38;region=BE&#38;gclid=Cj0KEQjwhN-6BRCJsePgxru9iIwBEiQAI8rq879lESeuIU4N4AEQRLNEXj4f8z65KkD-q8Xr35sQDRgaAp-k8P8HAQ&#160; </td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td> http://www.sigmaaldrich.com/catalog/product/sigma/g9023?lang=en&#38;region=BE&#160; </td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A-11034</td>
			<td> http://www.thermofisher.com/order/genome-database/searchResults?query=488&#38;resultPage=1&#38;resultsPerPage=15&#38;autocomplete=&#38;searchMode=keyword&#38;productTypeSelect=antibody&#38;targetTypeSelect=antibody_secondary&#38;species=ltechall&#38;keyword=488#filters=genericsort2:\%22Capra%20hircus\%22;;generic6:^\%22Oryctolagus%20cuniculus\%22$;;conjugates:^\%22Alexa%20Fluor&#38;reg;%20488\%22$;; </td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 594</td>
			<td>Thermo Scientific</td>
			<td>A-11032</td>
			<td> http://www.thermofisher.com/order/genome-database/searchResults?query=594&#38;resultPage=1&#38;resultsPerPage=15&#38;autocomplete=&#38;priorSearchTerms=&#38;searchMode=keyword&#38;productTypeSelect=antibody&#38;targetTypeSelect=antibody_secondary&#38;species=ltechall&#38;keyword=594#filters=genericsort2:\%22Capra%20hircus\%22;;generic6:^\%22Mus%20musculus\%22$;;&#160; </td>
		</tr>
		<tr>
			<td>VECTASHIELD mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td> http://vectorlabs.com/vectashield-mounting-medium-with-dapi.html&#160; </td>
		</tr>
		<tr>
			<td>DPBS, with calcium and magnesium</td>
			<td>Gibco</td>
			<td>14040174</td>
			<td> https://www.thermofisher.com/order/catalog/product/14040133&#160; </td>
		</tr>
	</tbody>
</table>

isolation of mouse endometrial epithelial and stromal cells for in vitro decidualization

蜕膜是子宫内膜间质细胞的孕酮依赖性分化过程，并且是成功的胚胎植入的先决条件。虽然已经进行了许多努力，以揭示蜕膜的基本机制，这是在与胚胎和底层基质细胞接触的上皮细胞之间的精确信令仍然知之甚少。因此，在一个同时的上皮细胞和间质细胞到能提高对蜕膜的分子细节的知识的方式学习蜕膜。为了这个目的， 在人工蜕膜的体内模型是生理最相关;然而，细胞间通信的操作受到限制。目前， 在子宫内膜间质细胞的体外培养物正在使用的几种信号分子，调查蜕膜的调制。通常，人或小鼠子宫内膜基质细胞用过的。然而，人样品的可用性经常受到限制。此外，使用鼠组织的是伴随着在培养的方法中的品种。这项研究提出了一个有效的和标准化的方法来获得纯子宫内膜上皮细胞（EEC），并使用用雌激素治疗，连续三天的成年小鼠完好基质细胞（ESC）的文化。该协议被优化以提高产量，活力，细胞的纯度和为了研究蜕膜中欧洲经济共同体和ESC的共培养，进一步延长。该模型可以是适合于利用在蜕膜两种细胞类型的重要性，并评估细胞间通信期间由EEC或ESC分泌显著信号分子的贡献。

协议的总体概述 图1A示出了协议的一般概述。每日注射E2（100毫微克），连续三天被用于同步动物和规范发情周期的阶段。动情周期可以通过阴道灌洗进行评估，并分为发情前期，发情，发情间期，并动情后期阶段。周期相可以根据在灌洗脱落细胞，它们经受荷尔蒙的变化的比例来指定。增加雌激素水平会使得动物进化到发情期。这个阶段可以容易地通过仅仅角化上皮细胞的存在和不存在的白细胞（ 图1B）来检测。在第4天，动物是在发情期的子宫被收集和MEEC和卓制隔离（ 图1C）。蜕膜ç一个通过在5天的孵化期加入0.5毫cAMP和1μM的MPA到2％FBS培养基来诱导。 MEEC和卓制文化的质量控制初级细胞培养物的纯度可以通过在波形蛋白和细胞角蛋白表达的差异进行评估。波形蛋白是间充质细胞中表达，因此，在子宫内膜间质细胞的中间丝。角蛋白是在上皮组织的细胞骨架中找到的中间丝。 图2A示出了使用QRT-PCR中MEEC和卓制波形蛋白和细胞角蛋白的mRNA表达水平进行评估。波形蛋白水平在卓制高和低MEEC（2.2倍高，P &lt;0.001），而细胞角蛋白水平在MEEC高和低卓制（高36倍，P &lt;0.001）。执行了波形/角蛋白双染，并指出，基质细胞表达波形蛋白，而epithelial细胞表达细胞角蛋白（ 图2B，C）。没有初级抗体被用作免疫染色（ 图2D，E）的阴性对照。这些免疫组织学染色显示，无论子宫内膜细胞培养物（MEEC和卓制）具有高达90％的纯度。 蜕膜在MEEC / MESC共培养分离后，小鼠子宫内膜癌和基质细胞的共培养系统接种以模仿内膜环境。细胞培养，直至上皮细胞中的细胞培养插管形成单层（800 TEER值 - 1,000 /孔），和间质细胞达到亚铺满状态。蜕膜是在基质细胞用0.5mM cAMP的应用和1μM的MPA诱导。温育后的五天，催乳素mRNA水平在所述基质细胞通过qRT-PCR评估作为decid的指示ualization（ 图3A）。催乳素水平在经受激素和检测不到与对照培养基（P &lt;0.01）温育的细胞的细胞中高表达。 由于上皮细胞是难以在细胞培养插入物内部可视化，生存力测定五天潜伏期（ 图3B）后立即进行。正常生长介质和存活力试剂的混合物加入到孔中，并孵育一段最小8 - 12小时。颜色从蓝色转变为明亮的粉红色表示可行的上皮细胞的存在。注意，当活细胞存在色移才会发生。 <img alt="图1" src="/files/ftp_upload/55168/55168fig1.jpg" /> 图1:协议的总体概述。 （A）开始的三个隔离协议方案雌激素注射的连续天。在第3天，进行必要的准备，应制备。在第4天，发情期通过执行阴道灌洗评价（由星号表示）。卓制和MEEC使用不同的消化步骤孤立。在第6天，或者当细胞汇合时，蜕膜可以在共培养系统中加入cAMP和MPA到介质和五天的潜伏期（ - 11 6天）诱导。 （B）中的发情期在阴道灌洗特征在于角化上皮细胞的存在的代表性图（放大率10X）。 （C）和卓制MEEC的代表图（放大率20X，比例尺:100微米）。 <a href="http://ecsource.jove.com/files/ftp_upload/55168/55168fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55168/55168fig2.jpg" /> <strong&gt;的图2:免疫染色和定量RT-PCR检测波形蛋白和细胞角蛋白在MEEC和卓制。 （ 一 ）波形蛋白和细胞角蛋白表达的信使RNA水平，表明文化的纯洁性。 RNA水平相对定量来持家基因ACTB和TBP的几何平均值。数据显示为平均值±SEM表示。上海卓制培养（B）和MEEC文化（C）波形/角蛋白双染。插入左侧显示了63X放大视图。阴性对照，省略了卓制（D）和MEEC（E）的第一抗体（比例尺:50微米）。 ***:P &lt;0.001与Bonferroni校正多个测试双向ANOVA。 <a href="http://ecsource.jove.com/files/ftp_upload/55168/55168fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:通过RT-PCR蜕膜验证。在间质细胞催乳素表达（A）mRNA水平来评价蜕膜。 RNA水平相对定量来持家基因PGK1和TBP的几何平均值。在控制或蜕膜培养基中培养的细胞的倍数变化表示为平均值±SEM表示。 （B）的前（上）和后（底）蜕膜刺激基质细胞的说明性图片。右边的插入物示出了蜕膜间质细胞的倍率。蜕膜的特征在于多核细胞的存在，由黑色箭头指示。 （C）的上皮细胞活力在测定后插入物评估的代表性图像。照片中的生存能力试剂的管理（Prestoblue）（0 h）和歼之后马上拍摄ollowing上午（ON）。 **:P &lt;0.01曼 - 惠特尼U检验。 ON:过夜。比例尺:100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55168/55168fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization at JoVE.com

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

这项研究提出了初级小鼠子宫内膜基质和上皮细胞的分离和培养，可以在一个共同培养系统中使用体外蜕膜研究的标准化和验证的方法。

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

isolation-mouse-endometrial-epithelial-stromal-cells-for-vitro

Microsoft Bing

Research

JoVE Journal

Developmental Biology

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

23.8K 次观看.  Katholieke Universiteit Leuven (KU Leuven). 这项研究提出了初级小鼠子宫内膜基质和上皮细胞的分离和培养，可以在一个共同培养系统中使用体外蜕膜研究的标准化和验证的方法。 

视频: Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

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 ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...