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. B., 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=Direct+reprogramming+of+human+neural+stem+cells+by+OCT4.">Direct reprogramming of human neural stem cells by OCT4.</a> Nature. 461, 649-643 (2009).</li><li>Totonchi, 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=Feeder-+and+serum-free+establishment+and+expansion+of+human+induced+pluripotent+stem+cells.">Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells.</a> Int. J. Dev. Biol. 54, 877-886 (2010).</li></ol>

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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。

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		<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>
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			<td>N-2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock: 100x</td>
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			<td>Albumin, from Bovine Serum</td>
			<td>Sigma</td>
			<td>A9418-50G</td>
			<td>Stock: 100%</td>
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			<td>Antibiotic - Antimycotic Solution</td>
			<td>Cellgro</td>
			<td>30-004-Cl</td>
			<td>Stock: Penicillin (10000 IU/ml); Streptomycin (10000 &#181;g/ml; Amphotericin (250&#181;g/ml)&#160;</td>
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			<td>DMEM&#160;&#160;</td>
			<td>Cellgro</td>
			<td>15-013-CV</td>
			<td></td>
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		<tr>
			<td>Precision Incubator Unit</td>
			<td>B.T.C. Engineering Milton Cambridge England</td>
			<td>Id.No. 840-374</td>
			<td></td>
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		<tr>
			<td>Glass Bottles for Rotating Unit</td>
			<td>B.T.C. Engineering</td>
			<td></td>
			<td></td>
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			<td>Silicone Rubber Cork</td>
			<td>B.T.C. Engineering</td>
			<td></td>
			<td></td>
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		<tr>
			<td>95% O2/5% CO2 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>
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		<tr>
			<td>Stemi SV6 Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 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>
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		<tr>
			<td>Weigh Balance</td>
			<td>Mettler Toledo&#160;</td>
			<td>AG285</td>
			<td></td>
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			<td>Centrifuge Tube &#8211; 50ml</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&#160;</td>
			<td>Roboz</td>
			<td>RS-6812</td>
			<td></td>
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			<td>Micro Dissecting Forceps &#8211; 4&#8221;</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Forceps - Hudson (cWALD) &#8211; 4-3/4&#8221;</td>
			<td>Roboz</td>
			<td>RS-5237</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (5/45)</td>
			<td>Roboz</td>
			<td>RS-5005</td>
			<td>Modified - Sharp ends were made blunt</td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (5)</td>
			<td>Roboz</td>
			<td>RS-5060</td>
			<td>Modified - Sharp ends were made blunt</td>
		</tr>
		<tr>
			<td>Micro Dissecting 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-100mm</td>
			<td>Fisher Scientific</td>
			<td>087571Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish-60mm</td>
			<td>Fisher Scientific</td>
			<td>0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish-35mm</td>
			<td>Fisher Scientific</td>
			<td>0875711YZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-150ml</td>
			<td>Corning</td>
			<td>431153</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-250ml</td>
			<td>Corning</td>
			<td>430756</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter System ( 0.22um Cellulose Acetate)-500ml</td>
			<td>Corning</td>
			<td>430758</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid Pipetter</td>
			<td>Drummond Scientific Co.</td>
			<td>D57849</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette-10ml</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serological Pipette-25ml</td>
			<td>Corning</td>
			<td>4251</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette - 7.7ml</td>
			<td>Thermo Scientific</td>
			<td>202-20S</td>
			<td></td>
		</tr>
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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

25.6K 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

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

科研

生物学

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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 ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

封闭的系统使用透氧材料科技HYPERStack船只的细胞培养协议

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.