Name: mouse embryonic development in a serum-free whole embryo culture system
Uploaded: 2014-03-01T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Mouse Embryonic Development in a Serum-free Whole Embryo Culture System at JoVE.com

我们要感谢朱莉戈登博士和南希·曼利博士为他们有益的建议与栽培技术。这项工作已经由儿童青光眼基金会和莎朗 - 斯图尔特无虹膜研究信托基金的支持。

小鼠胚胎培养： 所有试验程序都严格按照卫生指引，民族院校以下协议＃A2010 07-119，这是审查和批准格鲁吉亚实验动物管理和使用委员会，它保持持续监管的大学进行。 1。培养基的制备<ol><li>淘汰赛的DMEM，淘汰赛血清替代品（KSR）（10％），N-2补编（1X），白蛋白，从牛血清（2％），青霉素（：使用以下金额市售的干细胞培养基和补充准备培养基50国际单位/毫升），链霉素（50微克/毫升）和两性霉素-B（1.25微克/毫升）。制备的培养基是类似于先前描述的15做出了以下改变：除了抗真菌剂和使用的抗生素两倍的浓度（对于特定的试剂和设备的细节，请参见李材料ST）。 注：抗生素浓度与先前使用（摩尔-斯科特， 等[15]）是足够的胚胎培养进行长达24小时的时间段。然而，当继续培养超过24小时，我们经历了文化的污染，这是成功的抗生素浓度倍增的控制。 </li><li>储存媒体元件，在4°C或-20°C按照制造商的建议。 KSR和N-2补充应贮存在分装于-20℃，避免反复冻融。一旦打开淘汰赛的DMEM应在30天内予以确认按照制造商的建议。在使用之前，预热的水浴中的组分到37℃。 </li><li>准备介质在无菌条件下。消毒的所有设备和包含它们放置在培养罩在媒体面前组件用70％的酒精喷雾瓶的表面。 </li><li>媒体组件可以在无菌烧杯中，或者直接在过滤系统（Corning）中进行混合。首先，白蛋白粉末添加到淘汰赛的DMEM。通过轻轻晃动，直到白蛋白完全溶解调匀。然后加入N-2补充，KSR和抗生素，并用移液器调匀。然后过滤使用0.22微米孔径过滤器，真空吸消毒媒体。免除媒体到50ml锥形管，并储存在4℃直至使用。一旦准备的媒体应在4-5天培养使用。 </li></ol> 2。文化生产设备的研制<ol><li>消毒的玻璃培养瓶和橡胶瓶塞。包裹培养瓶在一个铝箔，并放置在橡胶瓶塞在水的烧杯中，并通过高压灭菌消毒。 </li><li>开始培养前用70％的酒精喷雾和温暖至37°C杀菌文化室（精密孵化器单位）。文化室为e打趣说一个会滚动的圆盘在其持有的培养瓶的中心。气体流进腔室由一个压力计和调节流率可以通过观察通过水在玻璃管中，在结束了气泡的流出进行调节。此滚动瓶培养装置是由新的和Cockroft 17引入的原始设备的修改版本。 </li><li>连接含有95％O 2/5％CO 2的混合气体在培养室中的气体钢瓶和调节气体流量为“每秒一个气泡”，这给出了50毫升/分钟的流速，并确保足够的氧供应向培养的胚胎。 </li></ol> 3。小鼠胚胎的收集和准备文化<ol><li>为了获得野生型小鼠胚胎中，我们利用C57BL/6J和CD-1（Charles River实验室）遗传背景品系的小鼠。 </li><li>检查雌性小鼠阴道塞，每天早上。在发现日当天中午Ë阴道塞应被视为E0.5 DPC（天性交后）。 </li><li>通过实施安乐死的孕鼠下列标准协议收集的小鼠胚胎在E10.5 DPC。小鼠利用CO 2吸入装置所指定的美国兽医协会（AVMA）准则动物的安乐死（2013）安乐死。以确保死亡，暴露于CO 2后进行颈椎脱位。 </li><li>喷上腹部腹面表面70％的乙醇，以避免腹毛粘到仪器。然后用锋利的钝操作剪刀和在microdissecting镊子来定位子宫一对4-3/4腹侧打开腹腔。使用4中microdissecting钳子抬起整个子宫，并通过在子宫体和在子宫角的切割尖端具有光操作剪刀它从主体中分离出来。 </li><li>赶紧用1X PBS漂洗整个子宫加热到37°C以除去任何血液附着于子宫，并立即在DMEM放置在培养皿中温热至37℃。进一步使用前用70％乙醇喷雾消毒器械。 注：预热的解决方案，包括PBS，DMEM培养液和培养基并在整个过程中保持它们在37℃的建议。 </li><li>在解剖显微镜下，子宫应当节段性解剖与轻作业剪刀。这将导致在上分段子宫，通过该一对改性（钝端）microdissecting镊子可以轻轻地插入，以扩大开口的任一侧上的小开口。这允许胎盘蜕膜，然后可以通过用一对microdissecting镊子轻轻撕开以露出顶叶卵黄囊（PYS）与赖克特的膜被除去的曝光。使用镊子的一个边缘沿着与PYS轻轻刺破赖克特的膜并从日分开Ë相关卵黄囊（VYS）层，露出胚胎完好VYS。的外胎盘锥和trophoectoderm衍生物可以与胎盘蜕膜或与赖克特的膜被去除。必须小心，以避免VYS破裂作为胚胎马上弹出。 </li><li>用完整VYS胚胎应被转移至培养皿含有用塑料移液管将培养基升温至37℃。 注：从子宫分离后，立即转移到培养液有助于获得更好的发展，文化的胚胎。 </li><li>一个小的开放应以卵黄囊有一对microdissecting镊子避免大血管。一个尖锐的镊子可以使用在相邻的头部区域的区域轻轻地刺入，以使所述开口。备选地两对钝镊子可以用来保持卵黄囊，轻轻撕，使一个小开口。然后，通过扩大与镊子大小刚好适合胚胎头轻轻扩大开放。羊膜被紧紧缠绕的胚胎应轻轻持远离身体与使用一对镊子的撕裂。胚胎头部和后来的全胚胎应轻轻地从卵黄囊，同时维持胚胎脉管系统与卵黄囊15的的完整性形象化。 注：卵黄囊血管的任何损坏都可能影响到文化的胚胎发育。受损的血管，例如胚胎不应被用于培养，并且可以在用于分段以后可以丢弃的胚胎。 </li><li>胚胎分期标准：检查解剖显微镜和组通过形态学标准包括身体和头部大小，四肢和眼的形态和阶段计数体节数（s）的各组至少两个胚胎在胚胎。 &lt;STRONG&gt;注：通常胚胎在子宫内的发育同一窝秀差异，并获得不同的车身尺寸，形态特征和体节数。然而，我们发现，胚胎由我们的形态学标准进行分组，通常具有相似的体节数（±1）。出于这个原因，它是不需要计数体节的每个胚胎，因为这会耽误时间，开始培养。 注：请不要使用被用来计数体节文化的胚胎。为了避免时延在开始培养开始培养对于其它胚后计数体节。被推迟到文化，从子宫分离后的胚胎通常表现出发育不良。 </li><li>立即胚​​胎转移到培养皿用新鲜培养基加热到37℃，并把它的文化引擎盖那里的胚胎应再次转移到培养皿中，用无菌文化视角e媒体并将其放入培养瓶中等之前启动的文化。 注意：对于胚胎的多个媒体传输之前培养有助于防止污染作为其中未安装在培养罩在解剖显微镜下进行的不同步骤胚胎收集和清扫。 注意：当产仔数大（&gt; 12个胚胎），进程一半的胚胎，并开始培养或把它们用在培养液中一盘，并将其放置在CO 2培养箱中在37°C当胚胎被留在外面的时间较长（&gt; 35-40分钟），我们观察到的心跳减缓，这将影响到文化的后期发展。 </li></ol> 4。培养小鼠胚胎<ol><li>在文化引擎盖打开蒸压培养瓶，妥善贴上标签。传输3 ml培养介质中加热到37°C至每吨他的瓶子，然后将小鼠胚胎应该轻轻地转移到使用无菌塑料移液管转移的培养瓶。在培养瓶然后应上铺橡胶瓶塞，这有助于保持培养瓶在培养装置的滚盘。 注意：该培养瓶与媒体可制备并放置在培养装置的轧制盘安乐死的小鼠前。这样就缩短了时间为转移的胚胎放入培养瓶。 注意：小鼠胚胎可以单独使用或作为共培养物在相同的培养瓶2或3的胚胎培养。 </li><li>培养瓶应在37℃下在无菌条件下进行的培养装置和紧钩他们在滚动圆盘与橡胶瓶塞的孔中。打开轧制盘以每分钟（rpm）35旋转而产生的恒定速度，这使得电磁制动的自由漂浮旋转在媒体上，也YOS由胚胎组织有助于游离气体交换。从连接到培养腔室中的气缸的气体流过滚盘和橡胶瓶塞入培养瓶。 </li><li>文化的胚胎16-40小时，并定期检查是否有气体流出和文化室的温度。 注：鼠标操纵胚胎培养：让胚胎得到适应的文化条件下至少30分钟。那么表现不佳，并显示微弱心跳的胚胎可以被丢弃，剩余的胚胎可用于进一步的处理研究。胚胎操作，如电穿孔5,9,16，显微注射18，胎圈注入16，药物治疗和其它程序可以在此时执行。我们成功地完成与某些信号分子治疗研究它们在早期眼睛发育的作用阿菲 - 凝胶琼脂糖珠植入。后的胚胎操作可以返回回来的新鲜培养基，继续培养。 </li><li> 9-10小时和18-19小时的文化时，继续培养40小时后更换培养基完全新鲜的媒体在特定的时间间隔。 注：可以不要求培养基更换，如果文化是由16〜18小时停止。 </li><li>在文化成功的措施：在培养胚胎存活率应可见心跳和血液循环在体内被确定，而在培养胚胎生长应该由增加的车身尺寸，体节数和头部，四肢，心脏和眼睛形态发育进行评估。 </li><li> 在子宫内的胚胎发展在E11.0 DPC（〜40-41次）和E12.0 DPC（〜49-50 s）应作为对照组进行比较胚胎16〜18小时和38-40小时，分别进行培养。 </li><li>在过程中培养和宫内阶段的不同时间点胚胎发育可CAPT解剖显微镜下ured。现货成像软件是用来捕捉图像，使用图像处理软件后处理。 </li></ol>

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P., 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=Estrogen+lowers+Alzheimer+beta-amyloid+generation+by+stimulating+trans-Golgi+network+vesicle+biogenesis.">Estrogen lowers Alzheimer beta-amyloid generation by stimulating trans-Golgi network vesicle biogenesis.</a> J. Biol. Chem. 277, 12128-12136 (2002).</li><li>Cheng, J., Dutra, A., Takesono, A., Garrett-Beal, L., Schwartzberg, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+generation+of+C57BL/6J+mouse+embryonic+stem+cells+in+a+defined+serum-free+media.">Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media.</a> Genesis. 39, 100-104 (2004).</li><li>Kim, J. 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妊娠中期阶段的小鼠胚胎在37℃下培养在无血清培养基中在95％O 2/5％CO 2中的滚动瓶培养装置的气氛中胚胎发育子宫外是极其依赖于多种因素，在每一步从子宫从安乐死的小鼠中分离的培养完成（ 图1）时的过程中。对影响发展的最重要因素是开始培养所需的时间。需要最关心的过程中的其它关键点包括以下步骤：如胚胎从子宫完好卵黄囊和从卵黄囊胚的外化的分离。由...

在体外利用全胚胎培养方法非常适合学习信号参与胚胎器官形成是否则难以在子宫内的访问机制。全胚胎提供组织的完整性，并支持适当的组织相互作用，这对于及时出现信令机制必不可少的关键对于器官在不同的细胞过程。而全胚胎培养物提供了大量的应用，例如移植的研究，基因和组织操作，珠子植入研究，毒理学研究等的平台。目前用于胚胎培养系统主要依赖于血清对胚胎的正常生长和维持在培养1-9。 血清已被用作主要的组件，从培养基中6-8，10，11的10-100％1。;然而，血清的组合物不能很好地定义，并且可以从不同动物对动物和每个时间的血清被收集。虽然实验室制备血清非常耗时，而且涉及到严格的程序，血清采购商业呈现相当大的差异在不同的地段和提高实验成本。添加到这些时，血清可含有未知因素，如生长因子，激素，或其他蛋白质，这可能会影响某些实验的结果，尤其是那些涉及信号传导分子的组织相互作用重要的研究。研究表明，除了血清对培养物可以潜在地改变某些信号分子的细胞内水平，如环磷酸腺苷（cAMP）和参与有丝分裂信号和磷酸肌醇3蛋白（PI 3） -激酶信号通路12-14。违背这些中，无血清培养系统提供的抗原的环境的优点，从生物活性酶，可以改变细胞过程和禁欲令各个实验的一致性。 在本研究中，我们使用由市售的干细胞培养基补充剂准备培养妊娠中期阶段整个胚胎在95％O 2/5％CO 2中的滚动瓶培养装置的气氛中的无血清培养基中，在37 °Ç15,16。小鼠胚胎根据这些规定条件16至40小时培养的进展表现在整体胚胎体和不同的结构，如心脏，四肢，大脑和眼睛的形态发展，指示相应的细胞增殖，迁移，分化和组织的互动水平。在培养的复杂器官系统，如眼睛中的一个的胚胎发育的分子分析显示眼发展为与在眼组织中观察到EMBR一致YOS发展在子宫内 （Kalaskar和代尔堡，在准备中）。因此，我们表明，在妊娠中期阶段培养的小鼠胚胎中，表现出进行性生长和形态的发展相媲美，在胚胎在子宫内的发展变化。

<table><tbody><tr><td>KnockOut DMEM</td><td>Invitrogen</td><td>10829-018</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>N-2 Supplement</td><td>Invitrogen</td><td>17502-048</td><td>Stock: 100x</td></tr><tr><td>Albumin, from Bovine Serum</td><td>Sigma</td><td>A9418-50G</td><td>Stock: 100%</td></tr><tr><td>Antibiotic - Antimycotic Solution</td><td>Cellgro</td><td>30-004-Cl</td><td>Stock: Penicillin (10,000 IU/ml); Streptomycin (10,000 &amp;micro;g/ml; Amphotericin (250 &amp;micro;g/ml)&amp;nbsp;</td></tr><tr><td>DMEM&amp;nbsp;&amp;nbsp;</td><td>Cellgro</td><td>15-013-CV</td><td></td></tr><tr><td>Precision Incubator Unit</td><td>B.T.C. Engineering Milton Cambridge England</td><td>Id.No. 840-374</td><td></td></tr><tr><td>Glass Bottles for Rotating Unit</td><td>B.T.C. Engineering</td><td></td><td></td></tr><tr><td>Silicone Rubber Cork</td><td>B.T.C. Engineering</td><td></td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt; Cylinder</td><td>AirGas Inc.</td><td></td><td></td></tr><tr><td>Stemi SV11 Apo Dissecting Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Stemi SV6 Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Water Jacketed Incubator</td><td>Forma Scientific</td><td>Model: 3110</td><td></td></tr><tr><td>Culture Hood</td><td>Nuaire Biological Safety Cabinets Class II TypeA2</td><td>Model: Nu-425-600</td><td></td></tr><tr><td>Water Bath</td><td>Fisher Scientific</td><td>IsoTemp205</td><td></td></tr><tr><td>Weigh Balance</td><td>Mettler Toledo</td><td>AG285</td><td></td></tr><tr><td>Centrifuge Tube, 50 ml</td><td>Corning</td><td>430291</td><td></td></tr><tr><td>Light Operating Scissors</td><td>Roboz</td><td>RS-6702</td><td></td></tr><tr><td>Operating Sharp-Blunt Scissors&amp;nbsp;</td><td>Roboz</td><td>RS-6812</td><td></td></tr><tr><td>Microdissecting Forceps, 4 in</td><td>Roboz</td><td>RS-5211</td><td></td></tr><tr><td>Microdissecting Forceps - Hudson (cWALD), 4-3/4 in</td><td>Roboz</td><td>RS-5237</td><td></td></tr><tr><td>Microdissecting Tweezers (5/45)</td><td>Roboz</td><td>RS-5005</td><td>Modified - Sharp ends were made blunt</td></tr><tr><td>Microdissecting Tweezers (5)</td><td>Roboz</td><td>RS-5060</td><td>Modified - Sharp ends were made blunt</td></tr><tr><td>Microdissecting Tweezers (55)</td><td>Roboz</td><td>RS-5063</td><td>Modified - Sharp ends were made blunt</td></tr><tr><td>Instrument Tray</td><td>Roboz</td><td>RT-1401S</td><td></td></tr><tr><td>Instrument Tray Lid</td><td>Roboz</td><td>RT-1401L</td><td></td></tr><tr><td>Petri Dish-100 mm</td><td>Fisher Scientific</td><td>087571Z</td><td></td></tr><tr><td>Petri Dish-60 mm</td><td>Fisher Scientific</td><td>0875713A</td><td></td></tr><tr><td>Petri Dish-35 mm</td><td>Fisher Scientific</td><td>0875711YZ</td><td></td></tr><tr><td>Filter System ( 0.22 &amp;micro;m Cellulose Acetate)-150 ml</td><td>Corning</td><td>431153</td><td></td></tr><tr><td>Filter System (0.22 &amp;micro;m Cellulose Acetate)-250 ml</td><td>Corning</td><td>430756</td><td></td></tr><tr><td>Filter System (0.22 &amp;micro;m Cellulose Acetate)-500 ml</td><td>Corning</td><td>430758</td><td></td></tr><tr><td>Pipet-aid Pipettor</td><td>Drummond Scientific Co.</td><td>D57849</td><td></td></tr><tr><td>Serological Pipette-10 ml</td><td>VWR</td><td>89130-898</td><td></td></tr><tr><td>Disposable Serological Pipette-25 ml</td><td>Corning</td><td>4251</td><td></td></tr><tr><td>Transfer Pipette - 7.7 ml</td><td>Thermo Scientific</td><td>202-20S</td><td></td></tr></tbody></table>

mouse embryonic development in a serum-free whole embryo culture system

妊娠中期阶段的小鼠胚胎进行培养，利用从在充氧轧制瓶培养系统可商购的干细胞培养基补充剂制备的无血清培养基中。小鼠胚胎在E10.5小心地从具有完好卵黄囊子宫分离出，并在涉及精确的外科手术操纵过程将胚胎从卵黄囊，同时保持胚胎的血管连续性卵黄囊轻轻形象化。相较于准备与完整卵黄囊或卵黄囊的胚胎取出，这些胚胎表现出卓越的成活率和类似条件下发育进程，当培养。我们表明，这些小鼠胚胎，在95％O 2/5％CO 2中的滚动瓶培养装置，在37℃下进行16-40小时的气氛中定义的培养基中培养时，表现出形态生长和发育媲美在胚胎发展在子宫内 。我们相信钍方法是将调查员需要利用全胚胎培养研究在胚胎器官形成的重要信令的交互非常有用。

小鼠胚胎的发育子宫外取决于从子宫从主体分离，以当时的胚胎进行培养的时间开始的多个因素。如描绘在图1中 ，该过程涉及一系列步骤，包括，妊娠子宫从主体分离（ 图1A），具有完整的卵黄囊（ 图1B）的胚胎的分离，从卵黄囊的胚外化（ 图1C），并在37℃下在95％O 2/5％CO 2中的滚动瓶培养装置的气氛下培养在无血清培养基中的...

Watch this Scientific Journal Video about Mouse Embryonic Development in a Serum-free Whole Embryo Culture System at JoVE.com

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

血清中胚培养利用含有能影响实验特别是在涉及信令交互研究的结果未知组分。在这里，我们采用无血清培养充氧系统和显示，妊娠中期小鼠胚胎为16-40小时培养的表现出形态学发展媲美的胚胎在子宫内的发展。

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 ...

mouse-embryonic-development-serum-free-whole-embryo-culture

Research

JoVE Journal

Biology

25.8K 次观看.  University of Georgia. 血清中胚培养利用含有能影响实验特别是在涉及信令交互研究的结果未知组分。在这里，我们采用无血清培养充氧系统和显示，妊娠中期小鼠胚胎为16-40小时培养的表现出形态学发展媲美的胚胎在子宫内的发展。 

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

科研

发育生物学

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

在小鼠全胚胎培养的神经管封闭

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

神经科学

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

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. ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

小鼠胚胎中早期心脏祖细胞的实时成像

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

在与 Ishikawa 细胞的体外共培养模型中评估胚胎粘附

Establishment of an Embryo Implantation Model In Vitro

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)&#160;preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-&#946;-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

作者聚焦:通过分析胚胎-子宫内膜相互作用推进提高妊娠率的治疗策略

This protocol describes methodologies to establish simple and efficient embryo implantation in vitro model for evaluating the relevant molecules affecting the embryo implantation process.

体外胚胎植入模型的建立

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

鼠胚体外发育无血清全胚胎培养系统

摘要

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此视频中的章节

小鼠胚胎干细胞的无血清单层培养神经细胞分化

在小鼠全胚胎培养的神经管封闭

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

文化早期鸡胚体外（新文化）的方法

基于微注射的免疫胚胎心肌细胞体内植入系统

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养

小鼠胚胎中早期心脏祖细胞的实时成像

大鼠血清适用于哺乳动物全胚胎培养的制备

体外胚胎植入模型的建立

鼠胚体外发育无血清全胚胎培养系统

摘要

探索更多视频

此视频中的章节

小鼠胚胎干细胞的无血清单层培养神经细胞分化

在小鼠全胚胎培养的神经管封闭

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

文化早期鸡胚体外（新文化）的方法

基于微注射的免疫胚胎心肌细胞体内植入系统

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

小鼠胚胎中早期心脏祖细胞的实时成像

大鼠血清适用于哺乳动物全胚胎培养的制备

体外胚胎植入模型的建立

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养