这项工作是按顺序一个NCCR前沿遗传学试验奖，青少年糖尿病研究基金会的资助41-2009-775和格兰特12-126875从侦探的frieForskningsråd/ Sundhed OG Sygdom资助。作者感谢SPAGNOLI实验室主办的视频拍摄。

该协议旨在扩大从鼠E10.5派生类器官胰腺上皮分离胰腺细胞。 该协议要求的伦理批准用于动物实验。 背胰芽1。解剖从E10.5小鼠胚胎<ol><li>牺牲定时妊娠小鼠胚胎一天（五）10.5，开有一对剪刀的腹部，取出两个子宫角，并放置在10厘米的培养皿中充满了冷磷酸盐缓冲液（PBS）或贝科改良基本培养基（DMEM）中保持在冰上。总的实验从祭祀细胞种植在60-90分钟做是为了防止细胞损伤。 </li><li>子宫分成用小剪刀个别胚胎段。转移一个胚胎为35 mm培养皿用冷PBS和可视化它在解剖显微镜从上面照射下。用解剖镊子取出周围的肌肉，蜕膜和蛋黄SAc和暴露胚胎。将胚胎放回冷DMEM培养基。胚胎可以很容易地使用3毫升塑料移液管/滴管转移。在继续之前隔离每个人胚胎以类似的方式。 </li><li>将胚胎放入干净35毫米培养皿中的PBS。 <ol><li>使用薄钳（0.05毫米宽），除去前肢。轻轻插入钳子的开口分离从脊髓区域的消化道（ 图1）。可选：为了更方便地看到肚子，取出胚胎的上身，下到下面的蛋黄秆心脏和尾部区域。 </li><li>找到胃，肝和肠。使用镊子，隔离从胃到肠的消化道，并将其放置在冷的DMEM在冰上（ 图1）。背胰芽附着背侧，后到胃（ 图1）。 </li><li>隔离在西米每个人胃肠道在继续之前拉尔方式。如果胚胎需要进行基因分型，收集的尾部在夹层的时间，并保持每一个个体胃肠道在不同的井在24孔板上用冰冷的DMEM中。 </li></ol></li><li>将一个胃肠道在一个干净的35毫米培养皿中冷PBS。现在使用的照明从下面的明视场的解剖显微镜下观察并剖析背胰芽。这些条件将优化卷的可视化。使用电解削尖钨针或20 G注射器针头，分离出胰芽用尽可能少的间充质尽可能周围上皮细胞（ 图1）。 <ol><li>传输的分离的芽到含有冷分散酶溶液（1.25毫克/毫升）为2-3分钟的培养皿中。从这点来说，传输可以最方便地与连接到一个口控制的柔性管火焰拉50微升玻璃毛细管进行。备选地，但更多的里失去了芽的SK，使用10微升自动移液器（的Pipetman）用适当的塑料头。 </li><li>在微观控制执行胰芽愿望和弹射。把胰芽早在PBS中。从使用的玻璃毛细管（ 图1）针和温柔的吸间质进一步清理隔离胰芽。 </li><li>当整个间质被删除，在冲洗冷PBS胰芽;在单井每个芽转移到冷的DMEM（60孔迷你托盘装满10微升冷的DMEM）。它不删除的间质中分散酶是重要的，这使得组织很粘。 </li></ol></li></ol> 2，电镀分散细胞与文化<ol><li>来自胚胎的火焰拉玻璃毛细管进60孔迷你托盘装满10微升PBS冲洗锥形井转让解剖上皮细胞。 <ol><li>转移芽成10微升胰蛋白酶0.05％的ND让它在37℃孵育4分钟。通过传送芽成井与10微升DMEM +10％胎牛血清（FCS）的灭活胰蛋白酶。 </li><li>通过皮薄毛细拉带吸管拉马吸离解细胞悬液。避免气泡在这个阶段，而上下吹打以解离的细胞是重要的。胰腺组织体最佳地小群体的5-15细胞开始，因此部分离解，建议（ 图1）。 </li></ol></li><li>普尔从几个胚胎细胞到Eppendorf管中，以尽量减少由于个体差异的处理。稀释细胞悬液冷冻基底膜以1:3的比例。等分的该混合物到96孔板中，8微升/孔，或在用于成像的最佳化的板（见下文）。 </li><li>将培养板在37℃下进行5分钟，使基底膜变厚。填补了井70微升的选择培养基（组织体或球体，看标签莱1和2），并在含5％CO 2和95％空气中于37℃的潮湿环境中留下</li><li>每一个第 4天更换培养基。每日监控的日益胰腺殖民地和成像记录下这一过程。 </li><li>感兴趣的小分子或蛋白质可以被添加到在该阶段的培养基为干扰实验，如先前报道28。 </li></ol> 表1：类器官培养基。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>材料名称</td><td>股票集中度</td><td>在决赛中浓度</td><td>的蓄积量</td></tr><tr><td>青霉素，链霉素</td><td> 100％ </td><td> 1％ </td><td> 50微升</td></tr><tr><td>淘汰赛血清替代（补充） </td><td> 100％ </td><td> 10％ </td><td> 500微升</td></tr><tr><td> 2 - 巯基乙醇</td><td> 14.3 M </TD&gt; <td> 0.1毫米</td><td> 1微升</td></tr><tr><td>佛波酯（PMA） </td><td> 16微米</td><td> 16纳米</td><td> 5微升</td></tr><tr><td> Y-27632（ROCK抑制剂） </td><td> 50毫米</td><td> 10微米</td><td> 1微升</td></tr><tr><td>表皮生长因子</td><td> 50微克/毫升</td><td> 25纳克/毫升</td><td> 2.5微升</td></tr><tr><td>重组人R-脊椎1 </td><td> 250微克/毫升</td><td> 500毫微克/毫升</td><td> 10微升</td></tr><tr><td> - 或 - </td><td></td><td></td><td></td></tr><tr><td>重组小鼠的R-脊椎蛋白1 </td><td> 250微克/毫升</td><td> 500毫微克/毫升</td><td> 10微升</td></tr><tr><td>重组人FGF1（的aFGF） </td><td> 100微克/毫升</td><td> 25微克/毫升</td><td> 1.25微升</td></tr><tr><td>肝素（Liquemin） </td><td> 2500 U / ml的</td><td> 2.5单位/毫升</td><td> 2微升</td></tr><tr><td>推荐binant人类FGF10 </td><td> 100微克/毫升</td><td> 100毫微克/毫升</td><td> 5微升</td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25微升</td></tr><tr><td>总</td><td></td><td></td><td> 5,000微升</td></tr></tbody></table> 表2：球体介质。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>材料名称</td><td>股票集中度</td><td>在决赛中浓度</td><td>的蓄积量</td></tr><tr><td>青霉素，链霉素</td><td> 100％ </td><td> 1％ </td><td> 50微升</td></tr><tr><td> B27×50（增刊） </td><td> 100％ </td><td> 10％ </td><td> 100微升</td></tr><tr><td>重组人FGF2（bFGF）的</td><td> 100微克/毫升</td><td> 64纳克/毫升</td><td> 3.2微升</td></tr><tr><td> Y-27632（ROCK抑制剂） </td><td> 50毫米</td><td> 10微米</td><td> 1微升</td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8微升</td></tr><tr><td>总</td><td></td><td></td><td> 5000微升</td></tr></tbody></table>类器官发展的进展3。成像<ol><li>图片类器官每天或通过时间推移显微镜用荧光延时显微镜。对于时间的推移成像，使用一个XY（Z）自动倒置荧光显微镜。 </li><li>存款小水滴的3μl，以4孔板或玻璃底板装有2-5毫升培养基。图像与10倍的远距离目标。注意：表达荧光示踪剂的转基因小鼠可以使用。电影1所示，例如组织体从的Pdx1，Ngn3的-ER TM-IRES-nGFP +小鼠4初始扩张。核GFP使用户能够跟踪的细胞作为单独的对象，但类似的原理可以应用到带膜荧光追踪细胞如作为MT / MG小鼠29。 </li><li>接种细胞，避免焦点漂移和设置软件控制自动化取1张/小时，3天或以上的在手动定义的位置后，开始时间推移成像3小时。对于每一个位置，获得一个微分干涉对比（DIC）的形象，以及对GFP信号，报告荧光标记物的表达。 </li></ol>组织体4。恢复的组织学<ol><li>放置在96孔板上的冰和除去培养基，用冰冷的PBS代替。这部分解聚基底膜。 </li><li>轻轻地吸出每个单独的组织体，为了不破坏整体架构使用了1000微升末端除去周围的基质胶。将每个类器官，以良好的用冰冷的PBS。置于冰上板。直接固定在基质胶也是可能的。 </li><li>修复类器官15分钟在4％多聚甲醛（PFA），冷冻保存在蔗糖和嵌入在明胶。处理每个类器官为cryosectioning和组织学如前所述（Johansson 等人 ，2007）4。 </li></ol> 5，恢复组织体进行PCR和生物化学<ol><li>放置在96孔板上的冰和除去介质。加入60微升每孔的RNAlater，以稳定和保护细胞的RNA。 </li><li>通过部分地在冰上解聚破坏它在每个孔的机械凝胶。无论是为了不破坏整体架构或者通过用200μl的前端机械地破坏凝胶恢复整个井（用基质胶）用1,000微升尖恢复单个组织体。使用1,000μl吸头的良好的内容转移到一个无RNA酶，无粘性的Eppendorf管放在冰上。 </li><li>用60微升的RNAlater洗井，其余内容添加到相同的离心管中。 </li><li>旋转的管子5分钟，在500〜1000×g离心在4℃下</li><li>去除上清，只留下20-30微升的RNAlater中的管连同粒料。对于生物化学，储存样本尽可能干燥。 </li><li>不要立即冻结在RNAlater样品;在4°CO / N店（允许RNAlater中彻底穿透组织）。该组织可以被储存在-20℃下长期贮存和以后可以对组织破坏和提取少量的RNA的处理。 </li></ol>

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The authors have nothing to disclose.

大规模生产的功能性β细胞在体外仍然无效1。在这个充满挑战的背景下，发育生物学的研究可能有助于破译所必需的功能性β细胞分化的确切信号。此协议允许在体外胚胎胰腺祖细胞的维持，扩展和分化。这包括形成产生胰岛素的β细胞的不共表达其他内分泌激素，有高水平的Pdx1的，表达了亲convertases的成熟胰岛素和有胰岛素处理28。系统内的重要的关键因素是FGF的活...

器官培养提供了桥梁在体内研究和复杂，但具有很强的针对性之间的差距细胞系模型的方便，但近似模拟一个有用的模型。在胰腺的情况下，不存在细胞系完全等价于胰腺祖细胞虽然有转化细胞系模拟内分泌和外分泌细胞。成人全胰腺不能培养;分离的内分泌胰岛可维持数周而不细胞增殖和组织切片可以保持在体外对几个小时5。胚胎胰腺培养已被广泛使用，不仅研究其发展，还探讨上皮-间充质相互作用4,6,7，对图像进行处理8或化学上与它们9干扰。两个器官培养方法主要应用于：第一种是在纤连蛋白包被的板2，它是型转换器中培养胰芽便于您进行成像的目的;第二个选项是文化在气-液界面3,4的最佳形态保留在过滤器上的器官。虽然非常有用，这些方法会导致一定程度的平坦化的;相比于正常发育和起始群体是复杂的，包括所有类型的胰腺细胞和间充质细胞的前体细胞的扩张是非常有限的。 的能力，培养和扩大分散的初级细胞是有价值的研究谱系关系，并发现分离的细胞类型10的固有性质。杉山等 11能保持胰腺祖细胞和内分泌祖细胞是保留了一些功能特点3-5天培养的饲养层。 Pancreatospheres，类似于神经球12和13微球体，已经扩大，从成人胰岛细胞和导管细胞虽然祖细胞的性质/干细胞产生这些球体是不明确的。另外，在与生理发育相反，pancreatospheres含有一些神经元14,15。领域也从最近胚胎胰腺祖细胞16,17和再生胰腺18具有良好的祖扩张和随后的分化产生，但未能概括形态。 从分散，通常被定义细胞自组织成微型器官3D模型最近已蓬勃发展，并模拟多个器官如肠道19,20，胃21，肝脏22，前列腺23和气管的开发或成年营业额24。在某些情况下，发育和形态发生分化已扼要重述在三维由ES细胞，因为是光学杯25，小肠26或脑27的情况。 在这里，我们DEScribe一个扩大分离的多能胰腺祖细胞在三维基质胶支架在那里他们可以分化和自我组织的方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

分离并接种于三维基质胶E10.5胰背祖概括胰腺发育。祖细胞可以最容易遵循的荧光记者。在我们的例子中，我们使用了转基因小鼠，表达不激活Neurog3 4（ 图2），在没有他莫昔芬，从而通过的Pdx1启动子（ 的Pdx1，Ngn3的-ER TM-nGFP）（电影1）控制的核GFP蛋白。 与组织体介质中，小簇细胞的初始压实发生在第一个小时。膨胀然后可以由簇在...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

在本协议中所述的三维培养方法概括胰腺发育从胚胎分散小鼠胰腺祖细胞，包括他们的大幅扩张，分化和形态发生成支器官。这种方法适合于成像，利基的功能的干扰和操纵。

体外胰腺器官从分散的小鼠胚胎祖细胞

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K 次观看.  Swiss Institute for Experimental Cancer Research. 在本协议中所述的三维培养方法概括胰腺发育从胚胎分散小鼠胰腺祖细胞，包括他们的大幅扩张，分化和形态发生成支器官。这种方法适合于成像，利基的功能的干扰和操纵。 