The authors were supported by: a CancerFree KIDS Research Grant, a March of Dimes Basil O&#8217;Connor Starter Scholar Award (#5-FY14-32), a Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Trustee Grant Award; a CCHMC Research Innovation and Pilot Funding Award; and CCHMC developmental funds. Authors also acknowledge the Capel laboratory for the initial optimization of this technique.

在这些研究中使用的所有小鼠是从Charles River实验室得到的CD-1小鼠。以前培养实验也已在其它菌株，如C57BL / 6J（数据未显示）进行的，但任何应变都可以使用。怀孕的成年女性约为2-3个月之久，并通过CO 2吸入颈椎脱位和前胚胎切除双侧开胸进行安乐死。小鼠饲养按照美国国立卫生研究院的指导方针，以及实验方案被批准由辛辛那提儿童医院医学中心的机构动物护理和使用委员会。 1.准备仪器仪表，文化传媒，和菜名<ol><li>制备一个70％的乙醇溶液。高压灭菌去离子水（用于制作加湿室以及制造/稀释溶液）。通过喷雾用70％的乙醇消毒清扫工具（钳子，剪刀，和27G的针头注射器）。 </li><li>备含有的1M钙和镁（80克NaCl，2克氯化钾，14.4克的 Na 2 HPO 4，2.4克KH 2 PO 4，6.75 ml的氯化钙 ，3.75毫升的1M MgCl 2的10X磷酸盐缓冲盐水（PBS） ）。搅拌使之溶解在1升无菌高压灭菌的水，然后过滤用滤纸。稀释10倍的水，使1×PBS。 </li><li>热灭活的胎牛血清（FBS），在55℃进行30分钟，并过滤消毒用0.22μm注射器过滤器。 </li><li>准备38个ml的1X完全培养基（称为完全DMEM，cDMEM），由Dulbecco改良的Eagle培养基（DMEM），10％的过滤青霉素 - 链霉素股票热灭活FBS和1/100稀释。这通常应使用新鲜，但是可以存储在4℃下1个月。 </li><li>每瓶VEGFR酪氨酸激酶抑制剂II（TKIⅡ）在DMSO为原料浓度为5mg / ml，且稀释库存与cDMEM使工作溶液。最后CON中心定位为这项研究是在cDMEM 1.875微克/毫升。根据该公司的建议，储备溶液是稳定长达6个月的，在-20℃下。至于工作的解决方案，使新鲜和在同一天使用。 注意:此药物在工作溶液的浓度应比所需的终浓度2倍高（因为它将稀释两倍以液滴）。与此同时，准备cDMEM含有DMSO中为"控制"处理相同体积相同体积。 </li><li>在蒸馏水中单独准备的尾部消化试剂（50毫米氢氧化钠和1M的Tris-HCl [pH值为8.0）。 </li><li>制作的XY PCR引物25μm的股票（XY正向5&#39;-TGA AGC TTT TGG CTT TGA G-3&#39;和XY逆向5&#39;-CCG CTG C​​CA AAT TCT TTG G-3&#39;）一起在无核酸酶水。 </li><li>准备50X TAE缓冲液（242克Trizma碱57.1冰醋酸，加入100毫升0.5M的EDTA，pH值8.5，加水至1升网络纳尔体积）。制备1X TAE电泳缓冲液（20 ml的50X TAE用水稀释至1L总体积）。 </li><li>要创建2.5％琼脂糖溶液（0.625克琼脂糖加入0.5ml 50X TAE缓冲24.5毫升的水），热琼脂糖溶液在微波炉直至沸腾，然后放凉至约55-65℃（能够触摸）。冷却后，加入2微升1％溴化乙锭溶液，倒入凝胶。 注意！溴化乙锭是一种诱变剂和致畸;请用丁腈手套和处理时应适当的安全协议。备选地，其他潜在毒性较低的凝胶拆解试剂或染料都可以使用。 </li><li>制备封闭液（PBS含有0.1％的Triton X-100，10％胎牛血清[FBS]，和3％牛血清白蛋白[BSA]），用于免疫荧光。 </li><li>制备用于免疫荧光洗液（PBS含有0.1％的Triton X-100，1％FBS，和3％BSA）中。 </li><li>制备PBTx（PBS含有0.1％的Triton X-100），用于免疫荧光。 </li><li>准备孵化室对Culture。补大（直径100mm）的培养皿用35ml高压灭菌的水。广场不远的地方清扫和文化将被执行。 </li></ol>来自小家鼠 2.隔离胎儿睾丸<ol><li>检查雌性小鼠的阴栓每天早晨。当天中午，在其阴道塞检测被认为是E0.5。安乐死的怀孕雌鼠在E11.5，与经批准的动物协议的方式。 </li><li>喷向下小鼠腹部用70％乙醇。 </li><li>打开腹部的皮肤和腹膜使用细剪刀V形切口拉回组织瓣，露出内部器官。 </li><li>定位在卵巢子宫角的两端。用剪刀切下卵巢和结缔组织分离含有从母体胚胎的子宫。 </li><li>轻轻切割胎盘相对的一侧，露出了卵黄囊打开子宫壁。要小心，不要PUncture卵黄囊，以避免损坏或丢失的胚胎。 </li><li>附近的胎盘切去除含胚胎卵黄囊，将它们放入含有PBS与钙和镁的一道菜。钙和镁离子的存在将有助于支持细胞粘附分子来维持组织架构和防止组织粘到清扫工具。 </li><li>用钳子，通过仔细穿刺卵黄囊创建其中胚可以滑出空穴除去卵黄囊和羊膜从胚胎。一旦胚胎是卵黄囊之外，剪断脐带。 </li><li>从现在开始，使用位于无菌组织培养罩显微镜。除去胚的头部，并通过与颈部的两侧镊子夹持丢弃。 </li><li>取出尾部并放入新的离心管XY PCR分析，以确定胚胎的性别。 注意:虽然它是最优的培养性腺尽快收获后，它也是POSsible去除尾部DNA和前培养执行XX / XY基因分型，以使每个胚的性别是已知的，只有所需的性需要培养。而基因型正在做，胚胎可以保持分开（以使它们能够被追踪）在4℃或在冰上直至准备培养。一个需要注意的是，这期间以外在子宫内或培养条件下培养过程中可能潜在地影响生存能力培养或后续开发。 </li><li>钉扎胎的腋下有一对钳（通常在弱手持镊子）固定在对培养皿底部的仰卧位的胚胎。保持这些钳到位，在整个解剖过程中仍然保持胚胎。 </li><li>与其他对镊子，去除皮肤覆盖腹部，轻轻取出肝，肠，和其他器官暴露背部体壁。 </li><li>作为性腺将位于背面壁体上的背主动脉的任一侧，沿着主体的中线运行的大血管，舀泌尿生殖脊封闭镊子下方并通过打开钳子和抬起除去泌尿生殖脊。由于性腺将被松散地贴在墙壁上，注意不要拉涨过快不删除这些连接或性腺可能会拉长，造成损害的器官。 </li><li>移动泌尿生殖嵴为cDMEM在培养皿中，以适应组织的媒体。 </li><li>使用27G的针头泌尿生殖嵴的其余部分分隔开性腺 - 中肾复杂。使用一个针向下压切，并使用其他正确引导所述组织，以允许最佳分离。避免使用对盘底部的锯切运动，锐利的针头会造成塑料碎片，能坚持到组织。请一定要保持完全附着于性腺中肾。 </li></ol> 3.培养性腺的具有小分子抑制剂<ol><li>将两个20μ升移液器每次15微升。切成约1-2毫米断使用干净，灭菌刀片为性腺和加法的控制cDMEM转移阻挡枪头之一，而另一个吸移管将仅用于药物处理cDMEM。 </li><li>排队性腺与他们平行的枪头长轴这样他们就可以很容易地使用截止尖吸管。小心性腺粘附在移液管尖的内部。 </li><li>标号一侧作为控制液滴和其它如含药微滴。只有两个液滴可容纳在35毫米盘的盖子。确保盖子上的标签匹配含尾组织的离心管中的标签。 </li><li>吸移管将15μlcDMEM的含有单个性腺成在小（35毫米）培养皿的盖的液滴（只使用盖，由于底部是太高，以适应加湿培养皿腔内）。将两个独立的含性腺液滴上的菜的两侧，以及分隔FROM彼此。检查显微镜下，以确保性腺已转移到液滴。 </li><li>到指定为控制所述液滴，增加额外的15微升cDMEM含有DMSO（在步骤1.5制），使用30微升总体积的液滴。仅使用"控制"吸管，不会接触毒品。 </li><li>的其他液滴（药物处理的样品）中，添加15微升含TKI-ⅡcDMEM，使用30微升总体积的液滴。只能使用指定药物处理的样品的吸管。 </li><li>使用吸管，摊开液滴在不断扩大的圆形图案，直到他们约15-18毫米，直径性腺位于大致在中间。定向所述性腺使得其位于在其一侧和性腺和中肾很容易分辨。定向所述肺，使得两个裂片平放。 <ol><li>虽然液滴直径可能会略有不同，确保性腺都没有莲花灯克和在适当位置由表面张力保持。确保液滴不相互接触或盖的一侧。如果没有表面张力，该机构将在培养过程中生长到盖和扁平化，从而扭曲器官的形态。 </li></ol></li><li>小心地将含微滴培养皿盖直立到水在大（100毫米）加湿器皿的表面上。确保没有气泡截留在盖的底部下方，并且它平放的水的表面上。不要反转盖子，注意不要让水接触到盖洗脱液滴位于顶部。 </li><li>要创建一个小的，潮湿的室内，应立即用较大的加湿器盘的盖中盖围住性腺文化。法尽快尽量减少任何媒体蒸发。确保更小的菜都有它和较大盘的盖子之间的空间自由走动;否则，冷凝可能使密封和b锁空气交换的组织。 </li><li>一旦两个小盖子被放置在室中，紧接在室放入孵化器。孵育液滴培养48小时，在湿润的 CO 2培养箱中在37℃。 </li></ol> 4.聚合酶链反应确定胚胎的性别<ol><li>添加胚胎尾部到1.5ml微量离心管的底部。 </li><li>添加到每个管200微升的50mM NaOH中。 </li><li>放置在95℃下15分钟，或直至组织完全溶解。 </li><li>加入50微升1M的Tris-HCl和旋涡轻轻简单。 </li><li>加入0.5μl25μM的引物溶液，2.5微升10X Taq缓冲液，0.5微升的10mM的dNTP（核苷酸），19.3微升核酸酶:在1.5ml微量离心管，通过每体积由样本的总数乘以一个最新的PCR主混合物免费的水，0.2微升DNA Taq聚合酶。拌匀。 </li><li>添加23微升PCR主混合物以PCR含有消化尾3微升裂解液管。 </li><li>轻弹管将它们混合，然后执行短的自旋使样品管的底部。 </li><li>运行XY（短）PCR程序（ 表1）。 </li><li>加载样品（与5倍的染料）和梯在1X TAE缓冲液在2.5％琼脂糖凝胶。 </li><li>运行琼脂糖凝胶电泳直到XX和XY波段可以解决。 </li><li>图像的紫外线和捕获图像的凝胶。 </li><li>分析的X染色体特异性VS Y染色体特异条带（X染色体= 331个碱基对[BP]和XY = 302基点）13; XY（男性）的样品将有331两个频段和302基点，而XX（女性）的样本将显示为一个单一的331 bp的条带。 </li></ol> 5.整装器官免疫第1天: <ol><li>使用1000微升吸管截止尖取下文化性腺。如果需要的话，它们可以是放置在PBS一碟洗掉培养基和试剂。放入PBS中于0.5 ml离心管。较小的管子尺寸将节约试剂和更容易地跟踪样本在管。 <ol><li>一定要保持控制和处理的样品，以及XX和XY样品，在不同的试管和文化与移液器单独设置删除它们，以避免潜在的药物污染。 </li></ol></li><li>用PBS洗两次性腺。从这点上，该协议可以使用1×PBS缺乏钙和镁。把它们洗干净，允许性腺沉到管（通过重力）的底部，然后除去上面的液体。小心不要吸取太靠近他们失去性腺。 </li><li>从管中除去尽可能多的PBS作为可能的。加入250微升4％多聚甲醛的PBS中含有0.1％的Triton X-100，和让孵育O / N在4℃下于一个摇杆。或者，该固定可以在4℃上的摇杆进行2小时。注意！ Paraformaldehyde是一种有毒物质，因此在处理时应遵守安全协议。 </li></ol>第2天: <ol start="4"><li>在250微升PBTx在室温两次冲洗性腺。 </li><li>用250μlPBTx，每次10分钟，在室温上的摇杆洗性腺3次。 </li><li>方框性腺至少1小时在250微升阻断在RT上的摇杆溶液。 </li><li>染色性腺O / N，在4℃在250微升的第一抗体稀释在封闭液摇臂。 </li></ol>第3天: <ol start="8"><li>在250微升洗液冲洗两次性腺。 </li><li>洗性腺3次用250μl洗涤液用于在每个摇杆10分钟，在室温。 </li><li>块中的性腺1小时的250微升阻断在RT上的摇杆解决方案。 </li><li>盖上离心管铝箔保护荧光的二抗避光。 </li><li>孵育性腺2-4小时，在RT上ARocker在稀释于封闭含赫斯特33342或者解决方案250微升二抗，摇臂上进行二次抗体和赫斯特33342孵化O / N在4℃。 </li><li>在250微升PBTx两次冲洗性腺。 </li><li>在250微升PBTx，每次10分钟，在室温上的摇杆洗性腺3次。 </li><li>使用截止的枪头，转移性腺到最小的液体幻灯片。去除多余的液体，吸液管，但是要确保组织不会干。 </li><li>东方在用钳子希望的方式性腺，赶紧加入一滴安装介质安装幻灯片上的性腺。确保安装介质大衣完全所有的样品;关键是要避免让样品干燥。 </li><li>适当大小使用盖玻片（1.5号方式），轻轻盖样品。让安装介质传播来联系盖玻片的整个表面上。如果有必要，非常轻按盖玻片，这样的角落有没有大的气泡。 </li><li>密封与周围的边缘清晰指甲油幻灯片降低安装介质的蒸发。商店安装载玻片在黑暗中在4℃。 </li></ol>

<ol>
	<li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+development+in+a+serum-free+whole+embryo+culture+system.">Mouse embryonic development in a serum-free whole embryo culture system.</a> J. Vis. Exp. (85), e50803 (2014).</li><li>Maatouk, D. M., 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=Stabilization+of+beta-catenin+in+XY+gonads+causes+male-to-female+sex-reversal.">Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal.</a> Hum. Mol. Genet. 17, 2949-2955 (2008).</li><li>DeFalco, T., Bhattacharya, I., Williams, A. V., Sams, D. M., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yolk-sac-derived+macrophages+regulate+fetal+testis+vascularization+and+morphogenesis.">Yolk-sac-derived macrophages regulate fetal testis vascularization and morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 111, E2384-E2393 (2014).</li><li>Coveney, D., Cool, J., Oliver, T., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ,+the+gonad.">Four-dimensional analysis of vascularization during primary development of an organ, the gonad.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7212-7217 (2008).</li><li>Walton, K. D., Kolterud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fetal+whole+intestine+culture+system+for+ex+vivo+manipulation+of+signaling+pathways+and+three-dimensional+live+imaging+of+villus+development.">Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.</a> J. Vis. Exp. (91), e51817 (2014).</li><li>Delmarcelle, A. S., Villacorte, M., Hick, A. C., Pierreux, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+culture+system+to+study+thyroid+development.">An ex vivo culture system to study thyroid development.</a> J. Vis. Exp. (88), e51641 (2014).</li><li>Costantini, F., Watanabe, T., Lu, B., Chi, X., Srinivas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+embryonic+mouse+kidney,+culture+in+vitro,+and+imaging+of+the+developing+organ.">Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ.</a> Cold Spring Harb. Protoc. 2011, (2011).</li><li>Petzold, K. M., Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+ex+vivo+culturing+of+embryonic+pancreas.">A system for ex vivo culturing of embryonic pancreas.</a> J. Vis. Exp. (66), e3979 (2012).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J. Vis. Exp. (51), e2720 (2011).</li><li>Combes, A. N., 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=et+al.Endothelial+cell+migration+directs+testis+cord+formation.">et al.Endothelial cell migration directs testis cord formation.</a> Dev. Biol. 326, 112-120 (2009).</li><li>Bott, R. C., McFee, R. M., Clopton, D. T., Toombs, C., Cupp, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+kinase+domain+region+receptor+are+involved+in+both+seminiferous+cord+formation+and+vascular+development+during+testis+morphogenesis+in+the+rat.">Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat.</a> Biol. Reprod. 75, 56-67 (2006).</li><li>Cool, J., DeFalco, T. J., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular-mesenchymal+cross-talk+through+Vegf+and+Pdgf+drives+organ+patterning.">Vascular-mesenchymal cross-talk through Vegf and Pdgf drives organ patterning.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 167-172 (2011).</li><li>Clapcote, S. J., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplex+PCR+assay+for+sex+determination+in+mice.">Simplex PCR assay for sex determination in mice.</a> BioTechniques. 38, 702, 704-706 (2005).</li><li>Nel-Themaat, L., 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=et+al.Morphometric+Analysis+of+Testis+Cord+Formation+in.">et al.Morphometric Analysis of Testis Cord Formation in.</a> Dev. Dyn. 238, 1100-1110 (2009).</li><li>Kent, J., Wheatley, S. C., Andrews, J. E., Sinclair, A. H., Koopman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+male-specific+role+for+SOX9+in+vertebrate+sex+determination.">A male-specific role for SOX9 in vertebrate sex determination.</a> Development. 122, 2813-2822 (1996).</li><li>Morais da Silva, S., 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=et+al.Sox9+expression+during+gonadal+development+implies+a+conserved+role+for+the+gene+in+testis+differentiation+in+mammals+and+birds.">et al.Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds.</a> 14, 62-68 (1996).</li><li>Rockich, B. 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=Sox9+plays+multiple+roles+in+the+lung+epithelium+during+branching+morphogenesis.">Sox9 plays multiple roles in the lung epithelium during branching morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 110, E4456-E4464 (2013).</li><li>Si-Tayeb, K., Lemaigre, F. P., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+and+development+of+the+liver.">Organogenesis and development of the liver.</a> Dev. Cell. 18, 175-189 (2010).</li><li>Brennan, J., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+tissue,+two+fates:+Molecular+genetic+events+that+underlie+testis+versus+ovary+development.">One tissue, two fates: Molecular genetic events that underlie testis versus ovary development.</a> Nat. Rev. Genet. 5, 509-521 (2004).</li><li>Martineau, J., Nordqvist, K., Tilmann, C., Lovell-Badge, R., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-specific+cell+migration+into+the+developing+gonad.">Male-specific cell migration into the developing gonad.</a> Curr. Biol. 7, 958-968 (1997).</li><li>Yokonishi, T., Sato, T., Katagiri, K., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+spermatogenesis+using+an+organ+culture+technique.">In vitro spermatogenesis using an organ culture technique.</a> Methods Mol. Biol. 927, 479-488 (2013).</li><li>Sato, T., 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=In+vitro+production+of+functional+sperm+in+cultured+neonatal+mouse+testes.">In vitro production of functional sperm in cultured neonatal mouse testes.</a> Nature. 471, 504-507 (2011).</li><li>Gohbara, A., 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=In+vitro+murine+spermatogenesis+in+an+organ+culture+system.">In vitro murine spermatogenesis in an organ culture system.</a> Biol. Reprod. 83, 261-267 (2010).</li></ol>

The authors declare that they have no competing financial interests.

这项研究表明，具有研究胎儿发育许多潜在的应用离体全器官液滴的方法。这种技术可以用于多种器官，和允许研究者解决生物学问题，是很难使用体内检查方法由于胚胎和潜在胚胎致死的交通不便。此培养方法比其他体外附加利益的方法，如哺乳动物细胞系:整个器官都可以使用，因此维持存在于子宫内临界细胞间的相互作用;而培养体积很小（约30微升），因此...

在体内在人类器官再生是非常有限的;因此，组织工程，组织和从由主机捐献单个细胞器官的发育，是变器官更换一个有吸引力的潜在疗法。但是，对于该治疗策略是成功的，因素和参与器官的形态形成的细胞相互作用，必须彻底的研究和易于理解的。由于无法研究特定器官与传统方法的发展，研究人员已转向替代全胚胎或整个器官培养。 Kalaskar 等人 1已经表明， 体外全胚胎培养得到类似的结果（在培养的胚胎的58％），以在子宫内发育，表明体外培养方法可用于器官发生研究一个可行的替代方案。 个性化的器官培养系统，例如本体外的dro诗人盖伊培养系统，允许对整个器官分析独立的全身效应，而通过加药理学试剂或抗体的允许操作的特定信号传导途径或细胞的相互作用。传统上，胎儿器官发育的研究已不限于转基因和基因敲除小鼠的技术中，除了递送母系药理试剂。但是，也有涉及这些技术和治疗方法在体内的技术问题;最关注围绕影响各种器官同时经常导致胚胎致死性的影响。研究操纵胎儿发育的另外一个关注的药理是药物对子宫内胚胎发育的母体效应（如药物产妇代谢之前到达胚胎），如果这样的试剂可以穿过胎盘屏障。 整个器官培养技术说明这里被改编自协议首先通过Maatouk 等人 2描述，其中整个胎儿性腺孵育在体外直立液滴培养物。培养的胎儿性腺之一显著优点是，小分子抑制剂能够通过简单的扩散容易地访问整个器官。 。DeFalco等人已经表明，利用在与小分子抑制剂结合本体外液滴培养方法可用于研究信令进程和性腺发育3期间发生相互作用;这些过程将难以检查体内由于技术上的挑战（例如，药物通过胎盘或影响使用基因或药理学方法多个器官杀伤力通路）。 液滴文化不仅在某些方面比在子宫内的实验改进，而且它是一个改进， 在体外和前六VO的系统。因为它们缺乏多样的细胞类型，缺乏临界外基质（ECM）组件，其允许器官结构的形成，并且可以在信号级联放大显示的工件的使用细胞系，研究形态发生是极为困难的。虽然组织工程在创造支架模拟ECM做显著的改善，对于缺乏知识的哪些信号需要通过器官中的细胞类型使得它具有挑战性的在体外建立一个器官系统。其它体外系统已被预先建立，研究器官，或更具体形态发生，并已为胎儿器官的琼脂4实时成像非常成功的，转孔5，过滤器6，和其他支架矩阵7,8。液滴培养系统的优点是，它允许形态的研究通过提供利用较少的试剂，其分别为邻的能力FTEN昂贵，而且还给予器官表面张力，这是生长和信令能力9重要。 在小鼠中，初始睾丸形态取胚胎（E）之间发生级E11.5和E13.5;这些阶段包括的最佳时间窗口用于检查影响性别特异性分化因子。间期间睾丸形成发生的关键过程是睾丸帘线结构的产生和睾丸特异性的血管网络的形成。利用这种体外全器官液滴培养系统，一个是能够改变雄性特异性血管化和通过使用小分子抑制剂的抑制睾丸形态发生阻断了受体的血管内皮生长因子（VEGF）的活性;血管内皮生长因子介导的血管重构是睾丸发育10-12的关键。这种技术可以成功地应用于其它器官，并且可以针对特定的时间发展的窗口。全安装器官成像允许重要结构的可视化以及来自各种抑制剂的给药引起的结构和细胞变化。重要的是，这个系统是在研究人员可以绕过从母体药品监督管理部门或系统中断期间体内靶基因战略的潜在混杂影响是有利的。因此，这整个器官离体液滴培养系统可以显著改善了解哪些胎儿发育过程中特别会出现特定的器官内的相互作用和信号的能力。

<table><tbody><tr><td>Superfrost Plus Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Cover Glasses: Squares (22 mm x 22 mm, No. 1.5)</td><td>Fisherbrand</td><td>12-541B</td><td></td></tr><tr><td>Sally Hansen Xtreme Wear Nail Polish, Invisible</td><td>Sally Hansen</td><td></td><td></td></tr><tr><td>8-Strip 0.2 ml PCR Tubes &amp;amp; Detached Flat Caps</td><td>GeneMate</td><td>T3218-1</td><td></td></tr><tr><td>Pipetman L P1,000L, P200L, P20L, P10L, P2L</td><td>Gilson</td><td>FA10006M, FA10005M, FA10003M, FA10002M, FA10001M</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>FST</td><td>91150-20</td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>91460-11</td><td></td></tr><tr><td>Posi-Click 1.7 ml microcentrifuge tubes</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Posi-Click 0.6 ml microcentrifuge tubes</td><td>Denville</td><td>C2176</td><td></td></tr><tr><td>10 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1096FR</td><td></td></tr><tr><td>20 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1121</td><td></td></tr><tr><td>200 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1122</td><td></td></tr><tr><td>1,000 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1126</td><td></td></tr><tr><td>1 ml syringe with 27 gauge needles</td><td>BD PrecisionGlide</td><td>309623</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>305559</td><td></td></tr><tr><td>0.2 &amp;mu;M PES syringe filter</td><td>VWR</td><td>28145-501</td><td></td></tr><tr><td>Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm</td><td>Whatman</td><td>1003-185</td><td></td></tr><tr><td>Primaria 35 mm Easy Grip Style Cell Culture Dish</td><td>Falcon/Corning</td><td>353801</td><td></td></tr><tr><td>Petri Dishes, Sterile (100 mm x 15 mm)</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>New Brunswick Galaxy 14 S CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Eppendorf</td><td>CO14S-120-0000</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Nuare</td><td>NU-425-400</td><td></td></tr><tr><td>Mini-centrifuge&amp;nbsp;</td><td>Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>BioExpress GyroMixer Variable XL</td><td>GeneMate</td><td>R-3200-1XL</td><td></td></tr><tr><td>Mastercycler Pro Thermal Cycler with control panel</td><td>Eppendorf</td><td>950040015</td><td></td></tr><tr><td>SMZ445 stereomicroscope</td><td>Nikon</td><td>SMZ445</td><td></td></tr><tr><td>MultiImage Light Cabinet with AlphaEase Software</td><td>Alpha Innotech Corporation</td><td>Discontinued</td><td></td></tr><tr><td>Absolute 200 proof Ethanol</td><td>Fisher</td><td>BP2818-500</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>Sodium Phosphate (Dibasic MW 142) Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S374-1</td><td></td></tr><tr><td>Potassium Phosphate (Monobasic MW 136) KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5379-1KG</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>S671-3</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911-1KG</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>M2393-100g</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C5670-100g</td><td></td></tr><tr><td>Ambion Nuclease-Free Water</td><td>Life Technologies</td><td>AM9938&amp;nbsp;</td><td></td></tr><tr><td>XY PCR Primer&amp;nbsp;</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher</td><td>A38-500</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA)</td><td>Fisher</td><td>BP2482-1</td><td></td></tr><tr><td>1% Ethidium bromide solution</td><td>Fisher</td><td>BP1302-10</td><td>Toxic</td></tr><tr><td>Agarose</td><td>GeneMate</td><td>E-3120-500</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>367176-2.5KG</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503-1KG</td><td></td></tr><tr><td>dNTP Set, 100 mM Solutions</td><td>Thermo Scientific</td><td>R0182</td><td></td></tr><tr><td>DNA Choice Taq polymerase with 10x Buffer</td><td>Denville</td><td>CB-4050-3</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>O4042-500</td><td>Toxic</td></tr><tr><td>FluorMount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Hydrogen Chloride (HCl)</td><td>Fisher</td><td>A144212</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation</td><td>Bioexpress</td><td>0332-100g</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), triple 100 nm filtered</td><td>Fisher</td><td>03-600-511</td><td>Heat-inactivate before using</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>Use at 1:100</td></tr><tr><td>Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>VEGFR Tyrosine Kinase Inhibitor II - CAS 269390-69-4 - Calbiochem</td><td>EMD Millipore</td><td>676481</td><td></td></tr><tr><td>Rabbit Anti-Sox9 Antibody</td><td>Millipore</td><td>AB5535</td><td>Use at dilution: 1:4,000</td></tr><tr><td>Rat Anti-Mouse PECAM1 (CD31) Antibody</td><td>BD Pharmingen</td><td>553370</td><td>Use at dilution: 1:250</td></tr><tr><td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td><td>Cell Signaling</td><td>9661S</td><td>Use at dilution: 1:250</td></tr><tr><td>Rat E-cadherin / CDH1 Antibody (ECCD-2)</td><td>Life Technologies</td><td>13-1900</td><td>Use at dilution: 1:500</td></tr><tr><td>Hoechst 3342, trihydrochloride, trihydrate</td><td>Invitrogen (Molecular Probes)</td><td>H1399</td><td>Use at 2 ug/ml</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>712-165-153</td><td>Use at dilution: 1:500</td></tr><tr><td>Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>712-605-153</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td><td>Life Technologies</td><td>A31572</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A21206</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A21208</td><td>Use at dilution: 1:500</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td><td>Life Technologies</td><td>A31573</td><td>Use at dilution: 1:500</td></tr></tbody></table>

using ex vivo upright droplet cultures of whole fetal organs to study developmental processes during mouse organogenesis

在子宫内调查器官是胎盘哺乳动物，由于试剂的交通不便在技术上具有挑战性的过程中对子宫内的发育胚胎。新开发的体外直立滴培养法提供了一个有吸引力的替代在子宫内进行的研究。 体外液滴文化提供了检查和处理，通过使用各种保护和活化化合物的细胞相互作用和多样化的信号通路的能力;另外地，可以研究在特定器官的发展多种药理试剂的作用，而不在子宫内的全身药物递送的不需要的副作用。相对于其他的体外系统中，微滴培养不仅允许研究三维形态发生和细胞-细胞相互作用的能力，这是不能在哺乳动物细胞系被再现，同时还要求显著少řeagents比其他离体和体外协议。本文示范了正确的小鼠胎儿器官解剖和直立液滴培养技术，其次是全器官免疫证明该方法的有效性。 体外液滴培养方法允许形成器官结构可比所观察到的体内 ，并且可以用来研究由于胚胎致死性在体内模型中 ，否则难以研究的过程。作为一个模型应用系统中，小分子抑制剂将用于探测血管的睾丸形态发生中的作用。此体外液滴培养方法是可扩展到其他胎儿器官系统，如肺和潜在别人，虽然每个器官必须是广泛的研究，以确定任何器官特异性修改的协议。这个器官培养系统提供了在实验与胎儿各器官的灵活性，并导致obtained。使用这种技术将有助于研究人员深入了解胎儿的发育。

体外培养液滴允许一个操纵整个器官，如生殖腺，来研究细胞相互作用和动力学。 图1展示了在一个逐步的方式如何准备E11.5性腺液滴培养。在培养协议的第一个步骤包括最初除去从母小鼠（图1A和1B）的含胚子宫。除去从母亲子宫后，子宫壁被切断，将胚胎从卵黄囊到PBS中用于进一步解剖（图1C-E）的解放。去除内脏器官的后?...

Watch this Scientific Journal Video about Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis at JoVE.com

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

using-ex-vivo-upright-droplet-cultures-whole-fetal-organs-to-study

Research

JoVE Journal

Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

12.4K 次观看.  Cincinnati Children's Hospital Medical Center. The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

视频: Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

科研

发育生物学

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

遗传学

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

免疫与感染

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

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

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

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 ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

生物学

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

神经科学

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

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此视频中的章节

器官培养和鼠标沃尔弗导管的全山免疫染色

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

人胎盘及蜕膜器官培养研究在母胎界面感染

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养

一个系统

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

鼠胚体外发育无血清全胚胎培养系统

运用

摘要

探索更多视频

此视频中的章节

器官培养和鼠标沃尔弗导管的全山免疫染色

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

人胎盘及蜕膜器官培养研究在母胎界面感染

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

一个系统

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

鼠胚体外发育无血清全胚胎培养系统

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养