我们感谢 Jolanta Chmielowiec 就该协议和手稿提供了有益的讨论。我们还感谢本杰明 Arenkiel 获得共聚焦显微镜。这项工作得到了 NIH (P30-DK079638 到手机和 T32HL092332-13 到 M.A.S. 和手机)、捷尔医学基金会 (手机) 以及在医疗知识和发展残疾研究中心的共焦核心 (nih U54 HD083092 的支持)。肯尼迪国家儿童健康和人类发展研究所)。

该方法所描述的所有动物实验均经贝勒医学院机构动物护理和使用委员会批准。
1. 小鼠胚胎日10.5 胰祖细胞的制备
注: 本协议不需要在无菌条件下遵循, 直到步骤 2, 但它是最佳的消毒解剖工具和喷雾与70% 乙醇之前使用。
<ol><li>为解剖, 填充冰桶和放置一个容器磷酸盐缓冲盐水 (PBS) 在冰。清洁两个细尖钳和解剖剪刀与70% 乙醇。设置在解剖区附近, 一个容器至少有三硼硅酸盐毛细管, 口吸管, 打火机, 过滤1000µL 吸管提示。<ol>
<li>准备至少1毫升的1.25 毫克/毫升冷酶稀释在消毒水和地方在第二井的12井板冰。将 PBS 放在12井板的第一、第三和第四口井中。在冰上放置1毫升0.05% 胰蛋白酶在1.5 毫升管。将震动孵化器预热至 37 oC。</li>
	</ol>
</li>
	<li>弄死 e10.5 符合 IACUC 准则的定时怀孕小鼠。用70% 乙醇喷雾腹部以清洁区域, 然后用剪刀和镊子在小鼠生殖器上做 V 形切口, 然后继续到隔膜。 注: 此协议中的阴道插头外观显示为0.5 天。使用 5-6 周大的老鼠, 体重增加超过2克的行为作为一个次要措施成功浸渍。<ol>
<li>通过在子宫最上部的一侧切开切口, 仔细切除子宫, 将器官从鼠标向上拉出, 然后沿着这个切口向下到生殖器区域, 然后再重复另一侧。把它放在一个10厘米培养皿与冷 PBS。 注意: 从 e11.5 分离细胞中获得 organoids 是可能的, 然而不同于 e10.5 pancreatoids, 这些 organoids 还没有被描绘出来, 因此可能无法保留区分成所有三种主要胰血统的能力。</li>
	</ol>
</li>
	<li>在光解剖显微镜下, 放置培养皿, 并小心地打开子宫组织通过放置两个钳在每个胚胎和剥离组织远离蛋黄囊。用镊子轻轻抓住蛋黄囊, 在10厘米培养皿中放入新鲜的冷 PBS, 转移胚胎。把培养皿放在冰上。 注意: 重要的是 heedfully 删除胚胎, 最好保持在蛋黄囊中, 因为强力去除可以拉脐带, 撕裂组织的胚胎和损害结构完整性。</li>
	<li>将多个 PBS 滴放在10厘米的培养皿盖上。使用这些水滴转移胚胎在解剖时, 胚外组织被删除, 以确保能见度。将胚胎转移到 pbs 上, 用镊子轻轻地从蛋黄囊中取出, 其余的胚胎留在 pbs 上 (图 1a)。把胚胎移到新的 PBS 上, 用镊子轻轻舀起胚, 不损伤组织 (图 1B)。 注意: 组织可能需要更频繁地转移到新鲜的 PBS 水滴比这里描述, 取决于液体的不透明度。<ol>
<li>在一个新的 PBS 下降, 删除前肢芽使用钳 (图 1B)。将镊子放入开口处, 肢体芽出现, 轻轻撕裂最外部组织前方 (图 1B)。旋转胚胎并在后方向重复, 停止在后肢芽。此时, 胃肠道应该可见 (图 1B)。</li>
		<li>胃肠道后区有轻微弯曲 (图 1F)。在胃肠道和体壁脊柱区之间的开口处插入镊子。分离胃肠道, 慢慢向上工作, 直到最前面的区域到达心脏区域连接的地方 (图 1B-E)。 可选: 从胃肠道的腹部切除原始心脏和肝芽, 只留下连续的肠道管。前几次这个协议是执行它可能会更容易离开心脏和肝脏的腹部标志性, 直到胃肠道和背胰芽的形态学更熟悉 (图 1C和D).</li>
		<li>将胃肠道转移到新鲜的 PBS 液滴上。</li>
		<li>取镊子, 轻轻捏起凸出芽和小肠下方的组织, 稍微抬起外层组织 (图 1d-F)。 注意: 背部的胰芽位于胃和肠道之间 (图 1c-G)。下面的胰芽应该看起来像一个圆形的, 诺比结构 (图1 G).</li>
		<li>放置钳, 芽连接到肠道和捏向上松开芽 (图 1G)。洗一次在一个新的 PBS 泡沫之前, 将芽转移到12井板的第一井, 在寒冷的 pbs 冰。重复直到所有芽从胚胎中收集, 并放置在12井板块的第一井。</li>
	</ol>
</li>
	<li>将12井盘放在光夹层显微镜下。计数成功解剖的胰芽的数量, 以后计算分裂比率。<ol>
<li>将过滤后的1000µL 吸管尖端放入口管中, 并附上毛细管。火焰对毛细管进行杀菌, 并在管子中产生弯曲, 便于使用。使用毛细管将芽转移到含有冷酶溶液的第二井, 2 分钟, 然后用干净的 PBS (图 1G) 向第三井传输。 注意: 将芽从酶转移到 PBS 时, 避免接触毛细血管管尖端的组织。如果组织被卡在管子的尖端, 吸管向上和向下或在干净的 PBS 溶液中摇动, 直到松开。</li>
		<li>吸管芽向上和向下, 并转移到第四井与清洁 PBS。最后, 使用毛细管装置转移芽到1.5 毫升管与0.05% 胰蛋白酶。将管置于预热后的 37 oC 振动筛中, 并在 1500 rpm 处晃动4分钟。</li>
		<li>漩涡为大约十年代然后立刻安置管子在离心机并且旋转5分钟在 200 g. 谨慎地删除所有但大约50µL 的解决方案, 以不打扰离心单元格 (图 1g)。</li>
	</ol>
</li>
</ol>2. 培养游离祖细胞形成 Pancreatoids
注: 使用标准的无菌程序时, 应在标准组织培养罩的无菌气氛下进行。
<ol><li>对于收集的每个芽, 请将400µL 器官介质16,17 (表 1) 添加到离心单元格中。脉冲涡三倍和板100µL 每井低附件96井板, 分裂芽在1:4 比率 (图 1G)。</li>
	<li>检查显微镜下以可视化分离的细胞自由浮动 (图 1H)。将养殖盘放在 37 oC 孵化器中, 以中等速度在摇杆上使用 5% CO2 。</li>
	<li>监控 pancreatoids 的进度。每3天更换100µL 的新鲜媒体, 仔细吹打媒体下的控制罩, 在离开 pancreatoid 在井中删除。另外, 新鲜的100µL 的介质可以添加到一个新的井和 pancreatoid 转移使用硼硅毛细管连接到嘴吸管和1000µL 过滤吸管提示。</li>
</ol>3. 荧光成像的处理 Pancreatoids
<ol><li>要处理 pancreatoids 的免疫荧光图像, 首先准备4% 多聚甲醛/PBS, pH7.4 (粉煤灰) 解决方案。在96井板井中放置50µL 4% 粉煤灰。<ol>
<li>采用硼硅酸盐毛细管和口吸管与1000µL 过滤吸管尖端, 转移 pancreatoids 从培养基尽可能少的培养基, 以尽量小的介质到新鲜的井与4% 粉煤灰。设置在摇杆在室温下15分钟。</li>
		<li>将 pancreatoids 从4% 粉煤灰输送到井中, 100 µL 的新鲜 pbs, 室温下的岩石 5-10 分钟. 重复共三个新鲜 pbs 洗涤。 注意: 该协议可以在这里暂停, pancreatoids 存储在 4 oC 的 PBS 中100µL, 最多一周。</li>
	</ol>
</li>
	<li>对于 wholemount 成像 (建议如果抗体是适当的, 在3.3 节的替代方法. 显微镜 3.4.), 从 pbs 转移到50µL 5% 驴血清在 1x PBS 与0.1% 非离子洗涤剂 (PBST) 阻拦。在 4 oC 或在室温下以4小时为一夜摇滚。<ol>
<li>将 pancreatoids 转移到一个新的含原抗体的井中, 在 1x PBST 中稀释50µL 5% 驴血清。优选地, 岩石为 24 h 在 4 oC (至少 12 h, 但至多 36 h)。 注: 在材料表中列出的抗体对 Chga (1:100)、DBA (1:400)、Vim (1:400) 和 Pdx1 (1:100) 适用于 wholemount 染色;其他抗体可能需要优化。</li>
		<li>将 pancreatoids 转移到含有100µL 新鲜 PBST 的井中, 在室温下清洗和摇动30分钟。重复这一步骤, 共三新鲜 PBST 洗涤。</li>
		<li>将 pancreatoids 转移到一个新的含二级抗体的井中, 在 1x PBST 中稀释50µL 5% 驴血清。在 4 oC 中保护板, 使其处于最佳的 24 h (至少12小时, 但高达36小时)。 注: 在材料表和 DAPI 核 counterstain 中列出的所有次级抗体均适用于 wholemount 染色。</li>
		<li>将 pancreatoids 转移到含有100µL 1x PBST 和岩石室温30分钟的新井中, 在 1x PBST 中重复三洗涤。在第三和最后的洗涤, 添加 300 nM DAPI。</li>
		<li>最后, 将100µL 的清洁 PBS 和存储在4个oC 上, 在通过共聚焦显微镜进行成像前长达一周的时间内进行保护, 不过最好的结果来自于染色后立即进行成像。为防止 pancreatoids 在成像过程中的移动, 但防止干燥, 将每个 pancreatoid 放入20µL 的 PBS 中。如果 pancreatoids 不保持静止, 则减少 PBS 音量。</li>
	</ol>
</li>
	<li>对于冰冻切片, 在 4 oC 上隔夜将 pancreatoids 从 PBS 转移到30% 蔗糖溶液中。<ol>
<li>在夹层显微镜下的气泡中添加嵌入介质。在显微控制下使用镊子, 通过在嵌入介质中轻轻地用力挤压 pancreatoids, 将残留蔗糖去除, 然后再用冷冻嵌入介质转移到组织块。</li>
		<li>将组织块放在干冰上, 直到冷冻, 恒温器在8µm 的部分。 注意: 节可以立即存储在-80 oC 或 immunostained 中。</li>
		<li>允许从-80 oC 中的节在室温下解冻约5分钟, 直到干燥。使用疏水性的 pap 笔在组织和块使用5% 驴血清稀释 1x PBST 30 分钟在室温下, 绘制疏水性屏障。</li>
		<li>在 4 oC 中, 在 1x PBST 的一夜之间, 用5% 驴血清稀释的原抗体去除介质并立即更换。</li>
		<li>取出主要溶液, 用 1x PBST 三次, 每10分钟清洗组织。在此之后, 添加二次抗体溶液稀释5% 驴血清稀释在 1x PBST 1 小时的室温和保护幻灯片免受光照。</li>
		<li>删除第二个解决方案, 并在 1x PBST 中清洗幻灯片三次, 每10分钟。在最后的洗涤, 添加 300 nM DAPI。</li>
		<li>删除介质并添加安装介质和盖玻片。在 4 oC 中存储幻灯片, 直到准备好映像。</li>
	</ol>
</li>
</ol>4. 从 Pancreatoids 中分离 RNA 进行转录分析
<ol><li>收集 pancreatoids 使用含硼硅酸盐毛细管与过滤1000µL 吸管提示不育, 并放入500µL 酸胍硫氰酸盐-苯酚-氯仿溶液在1.5 毫升管。存储在-80 oC 或立即进行 RNA 隔离。</li>
	<li>对于 RNA 分离, 在冰上解冻样品, 如有必要, 加入100µL 氯仿。漩涡井和地方在 4 oc 为15分钟前冷却离心机到 4 oc。<ol>
<li>将管子放入离心机中, 在 4 oC 的1.2万克处旋转20分钟。</li>
		<li>小心移除管子, 以免干扰层的分离。使用200µL 吸管, 收集明确的水溶液, 并放置到一个新的1.5 毫升管, 留下少量的缓冲从白色蛋白层和粉红色的 DNA 层。不要触及这个过程中的任何东西的吸管的尖端, 不要触摸1.5 毫升管的墙壁。</li>
		<li>添加500µL 100% 异丙醇的新管含有水层和漩涡。让我们在室温下坐20分钟, 在冰上再长一点, 或者在-20 oC 上过夜的最大降水。</li>
		<li>离心机以1.2万克为15分钟, 在 4 oC 上颗粒状 RNA。除去异丙醇, 不干扰颗粒。 注: 由于 pancreatoids 小, 很可能颗粒不会被看到。</li>
		<li>加入冷, 75% 乙醇和反转管几次。放置在离心机和旋转在 9000 x g 10 分钟在 4 oc 去除乙醇和旋转2分钟在 9000 x g。</li>
		<li>使用200µL 吸管去除管中残留的乙醇, 而不接触颗粒所在的区域。在室温下将瓶盖打开 5-10 分钟, 以确保剩余乙醇的蒸发。</li>
		<li>并用重悬颗粒由涡流在20µL 清洁, 核酸酶水。 可选: 将 RNA 加热 65 oC 以3分钟, 然后立即返回到冰上。RNA 可以存储在-80 oC 或立即用于反向转录和 qPCR。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

细胞培养模型的进展对于正确的模型开发, 产生临床相关的细胞类型, 测试药物疗效, 甚至移植到病人来说是至关重要的。然而, 人工综述在菜肴中的发展是有挑战性的, 因为我们还远未了解器官和生理的机制在体内.因此,体外细胞的生成效率低下, 不能充分发挥作用, 不能长时间维持, 或使体内可比细胞的其他异常发生。这是因为许多不同的细胞类型相互作用, 而复杂的形态发生变化影?...

界定正常发展和生理学的机制, 对于了解疾病病因, 最终培养治疗方法至关重要。虽然培养和区分干细胞能够快速和高通量地分析发展, 但它受现有的知识机构的限制, 这些机制调控细胞命运和人为概括发展在一个相对同构, 二维状态1,2。不仅是受外部影响影响的 "体内" 开发, 在利基和环境中提供分泌信号和组织支持的不同单元格类型也可用于引导器官, 但这些细胞的功能也依赖于其用于指导的环境3,4,5。鉴于这些外部线索的重要性, 差异化协议的局限性, 以及体内老鼠模型的费力性质, 需要新的系统来实验研究基本的发育过程和生理学。
生成三维复杂 organoids 的协议的出现, 提供了一个方便和一致的系统来研究器官发生、生理学、药物功效, 甚至发病机制.建立小鼠 organoids 为不同的系统, 如胃6和肠道7已经扩大了我们对器官的理解, 提供了一个工具来研究发展复杂性较少的限制比在体内和体外模型。由于这些进展在小鼠 organoid 形成和人类多潜能干细胞的出现, 人类肠道8, 视网膜9, 肾脏10,11, 和大脑12 organoids 已经产生, 这《汇辑》仅限于现有关于发展机制的知识。
特别感兴趣的是胰腺 organoids 的产生, 因为无数的疾病瘟疫不同的胰腺细胞类型, 包括腺泡细胞和导管在外分泌的胰腺功能不全13, 胰腺炎的腺泡细胞14, 并β细胞在糖尿病15。获得有关这些不同细胞类型发展的知识有助于理解其病理学, 也可以作为个性化药物筛选或移植的平台。以前, Greggio et开发了一种方法来创建小鼠胰腺 organoids, 它概括了体内形态发生并发展了由所有主要的胰腺上皮细胞组成的三维复杂结构。类型16,17。这是胰腺领域向前迈出的重要一步, 特别是使细胞体外能够使β细胞的生物研究得以发展。然而, 在本议定书中形成的内分泌细胞的匮乏, 除非 organoids 移植到组织, 在那里利基可以互动和提供指导提示17。间质构成利基的最大部分, 严重包裹发育上皮从器官的早期阶段到以后阶段, 包括内分泌分层和分化 3, 4, 18。间质与发展中的胰腺的相互作用又是外部信号的另一个例子, 也是维持体内细胞复杂性来研究器官的重要性. 
在这里, 我们描述如何产生三维胰腺 organoids, 称为 pancreatoids, 从分离 e10.5 小鼠胰腺祖细胞。这些 pancreatoids 保留本机间质, 自组装在自由漂浮条件, 并产生所有主要的胰腺细胞类型, 包括强健的数量的内分泌β细胞 19.这种方法最适合分析内分泌的发展, 因为以前的协议缺乏强健的内分泌分化。然而, 使用 Greggio et所描述的胰 organoids 的协议, 更适合于分析胰腺上皮分支和形态发生, 因为 pancreatoids 中分支更受限制。

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td></td></tr><tr><td>BarnStead NanoPure Nuclease-free water</td><td>ThermoFisher</td><td>D119</td><td></td></tr><tr><td>Borosilicate Capillary Tubes</td><td>Sutter Instruments</td><td>GB1007515</td><td>O.D. 1mm, I.D. 0.75mm, 1.5cm length</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>C5080</td><td></td></tr><tr><td>Cell-Repellent 96-Well Microplate</td><td>Greiner Bio-One</td><td>650970</td><td>U-bottom</td></tr><tr><td>Centrifuge 5424 R</td><td>Eppendorf</td><td>5401000013</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>233306</td><td></td></tr><tr><td>Chromogranin-A antibody</td><td>Abcam</td><td>ab15160</td><td></td></tr><tr><td>Compact, Modular Stereo Microscope M60</td><td>Leica</td><td></td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10310</td><td></td></tr><tr><td>Countess Cell Counter Slides</td><td>Invitrogen</td><td>C10312</td><td></td></tr><tr><td>CryoStar NX70</td><td>ThermoFisher</td><td>957000L</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidine-2&#039;-phenylindole-dihydrochloride)</td><td>Roche</td><td>10 236 276 001</td><td>Powder</td></tr><tr><td>DBA antibody</td><td>Vector Lab</td><td>RL-1032</td><td></td></tr><tr><td>Dispase II, Powder</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>DMEM/F-12, HEPES</td><td>Gibco</td><td>11330032</td><td></td></tr><tr><td>Dnase I</td><td>Invitrogen</td><td>18068-015</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>0.05 x 0.02 mm; Titanium; Biology tip</td></tr><tr><td>EGF (Epidermal growth factor)</td><td>Sigma-Aldrich</td><td>E9644</td><td></td></tr><tr><td>Ethanol, 200 Proof</td><td>Decon Laboratories</td><td>2716</td><td></td></tr><tr><td>Forma Steri Cycle CO2 Incubators</td><td>ThermoFisher</td><td>370</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>OB10001</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td></td></tr><tr><td>INSM1 Antibody</td><td>Santa Cruz BioTechnology</td><td>sc-271408</td><td>Polyclonal Mouse IgG</td></tr><tr><td>Isopropanol</td><td>Fisher</td><td>a4164</td><td></td></tr><tr><td>Isothesia Isoflurane, USP</td><td>Henry Schein</td><td>11695-6776-2</td><td></td></tr><tr><td>Insulin Antibody</td><td>Dako</td><td>A056401</td><td>Polyclonal Guinea Pig</td></tr><tr><td>KAPA SYBR FAST Universal</td><td>KAPA Biosystems</td><td>KK4618</td><td></td></tr><tr><td>KCl</td><td>KaryoMax</td><td>10575090</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>Invitrogen</td><td>10828028</td><td></td></tr><tr><td>Leica TCS SPE High-Resolution Spectral Confocal</td><td>Leica</td><td></td><td></td></tr><tr><td>MgCl2</td><td>Sigma-Aldrich</td><td>442615</td><td></td></tr><tr><td>Mouse C-Peptide ELISA</td><td>ALPCO</td><td>80-CPTMS-E01</td><td></td></tr><tr><td>Mouse Ultrasensitive Insulin ELISA</td><td>ALPCO</td><td>80-INSMSU-E01</td><td></td></tr><tr><td>MX35 Microtome Blades</td><td>ThermoFisher</td><td>3052835</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>NaHCO3</td><td>Sigma-Aldrich</td><td>S3817</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>PBS 1X</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Pdx1 antibody</td><td>DSHB</td><td>F6A11</td><td>Monoclonal Mouse MIgG1</td></tr><tr><td>Peel-A-Way Disposable Embedding Molds</td><td>VWR</td><td>15160-157</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Corning</td><td>MT30002CI</td><td></td></tr><tr><td>PMA (Phorbol 12-Myristate 13-Acetate)</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Protein LoBind Microcentrifuge Tubes</td><td>Eppendorf</td><td>22431081</td><td>1.5mL Capacity</td></tr><tr><td>Recombinant Human FGF-10 Protein</td><td>R&amp;amp;D Systems</td><td>345-FG</td><td></td></tr><tr><td>Recombinant Human FGF-Acidic</td><td>Peprotech</td><td>100-17A</td><td></td></tr><tr><td>Recombinant Human R-Spondin I Protein</td><td>R&amp;amp;D Systems</td><td>4546-RS</td><td></td></tr><tr><td>BenchRocker 2D</td><td>Benchmark</td><td>BR2000</td><td></td></tr><tr><td>Sucrose 500g</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>SuperFrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Super Pap Pen</td><td>Electron Microscopy Sciences</td><td>71310</td><td></td></tr><tr><td>Thermomixer R</td><td>Eppendorf</td><td>05-412-401</td><td></td></tr><tr><td>Tissue Tek O.C.T. Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>TrypLE Express</td><td>Invitrogen</td><td>12604039</td><td>(1x), no Phenol Red</td></tr><tr><td>Trypan Blue Stain</td><td>Invitrogen</td><td>15250061</td><td>For cell counting slides</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200072</td><td>Phenol Red</td></tr><tr><td>Ultra-Low Attachment 24-Well Plate</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>Ultra-Low Attachment Spheroid Plate 96-Well</td><td>Corning</td><td>4520</td><td></td></tr><tr><td>Vimentin Antibody</td><td>EMD Millipore</td><td>AB5733</td><td>Polyclonal Chicken IgY</td></tr><tr><td>Vortex Genie</td><td>BioExpres</td><td>S-7350-1</td><td></td></tr><tr><td>Y-27632 Dihydrochloride</td><td>R&amp;amp;D Systems</td><td>1254</td><td>Also known as ROCK inhibitor</td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro

胰腺是由许多不同细胞类型组成的复杂器官, 它们协同工作来调节血糖的稳态和消化。这些细胞类型包括分泌酶的腺泡细胞, 一种 arborized 导管系统, 负责将酶输送到肠道, 以及产生激素的内分泌细胞。
内分泌β细胞是体内唯一的细胞类型, 它能产生胰岛素以降低血糖水平。糖尿病是一种以丧失或β细胞功能障碍为特征的疾病, 正达到流行的程度。因此, 必须建立一些协议来调查β细胞的发展, 它可以用于筛选目的, 以获得药物和细胞的治疗方法。虽然实验性研究老鼠的发展是必不可少的,在体内研究是费力和耗时的。培养细胞为筛查提供了更方便的平台;然而, 他们无法维持细胞多样性, 建筑组织, 和细胞之间的相互作用发现在体内.因此, 有必要开发新的工具来调查胰腺器官和生理。
胰腺上皮细胞在与间质的密切联系中发展, 从器官发生开始, 细胞组织并分化成复杂的、生理上有能力的成体器官。胰腺间质为内分泌的发展提供了重要信号, 其中许多还没有得到很好的理解, 因此在体外文化中很难重述。在这里, 我们描述一个协议, 文化三维, 细胞复杂的老鼠 organoids 保留间质, 称为 pancreatoids。e10.5 小鼠胰芽被解剖, 分离, 并培养在一个无支架的环境。这些漂浮细胞自组装与间质包络的发展 pancreatoid 和强健的数量的内分泌β细胞与腺泡和导管细胞一起发展。该系统可用于研究细胞的命运决定, 结构组织, 和形态发生, 细胞间的相互作用, 在器官发生, 或为药物, 小分子, 或基因筛选。

仔细解剖小鼠胚胎在 e10.5 从子宫角应该产生未损坏的胚胎在 PBS 进一步解剖 (图 1A)。胃肠道可以有效地从胚胎中移除 (图 1B), 允许辨别肠道和胃交界处的背胰芽 (图 1C-F)。e10.5 的胰芽以前有特征;祖祖先应表达Pdx1、Sox9、Ptf1a、和Hes120?...

Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

generation-scaffold-free-three-dimensional-insulin-expressing

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

9.0K 次观看.  Baylor College of Medicine. 在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。

视频: Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

科研

发育生物学

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工程

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

生物学

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

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 ...

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.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

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 ...

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

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