We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

注:所有小鼠，采用经美国密歇根医学院单位的大学实验动物医学根据大学委员会关于利用动物和护理的指导方针的协议处理人道。 1，全器官培养系统<ol><li>传媒与文化板块的准备<ol><li>在组织培养罩，从股市瓶取出5毫升BGJb媒体加入5 ml青霉素/链霉素的。准备媒体工作的股票在50毫升离心管中，加入1毫升5毫克/毫升抗坏血酸49毫升BGJb媒体（含青霉素/链霉素）。 </li><li>加入1 ml下面各转孔的准备了6孔培养板。 注意:如果不使用所有的6转孔，将多余的转孔进入一个新的无菌6孔培养板，供以后使用。再加上工作BGJb媒体每三个10厘米无菌培养皿中3毫升。 </li></ol></li><li>解剖准备<ol><li>浸泡解剖工具，70％ETHA北环线，一定要保持清洁工具在工作时。 </li><li>设置了一个大冰桶的解剖区域，并放置在6孔培养板和10毫米培养皿中与BGJb介质上的冰。冰一样多，而解剖和准备肠道对文化工作成为可能。 </li><li>麻醉的成年雌性小鼠与异氟烷（100微升）之前安乐死颈椎脱位胎儿肠道的收获室。 </li><li>之后，从他们的母亲收获的胎儿，舞台仔细按照Theiler分期图表13每胎。不要依赖于天后性交，以确定每胎为最多24小时的发育阶段变化的时代中的一个垃圾例行观察。 </li></ol></li><li>胎儿肠道的解剖<ol><li>之前去除肠子安乐死胎儿被斩首。 </li><li>解剖在1×Dulbecco氏磷酸盐缓冲盐水（DPBS）各肠然后PLACE成10cm的培养皿与工作BGJb媒体。 </li><li>小心地从胃健脾和胃和十二指肠上部的胰腺。 </li><li>轻轻分开网膜照顾到离开附尽可能和分离的连接仅足够大网膜，以允许在肠道被拉直。 注:对于比E14.5老年人或那些被培养超过24小时，肠，轻轻分开肠道分成3个相等的段，以允许腔内容物的流动通过肠和从切割端排出。然而，对于培养开始之前，E13.5，等待培养直到24小时后分离出肠的3个区段，以使浆膜到正常生长在肠的长度之前。 </li><li>用吸管口在肠道件转移到培养皿（1.1.2）的转孔。 </li><li>根据需要，使它们径直穿过transwel轻轻地重新定位肠升。 注意:一旦肠道开始通过管道移动管腔内容，在肠道内的任何扭结会导致备份和损害肠道。 </li></ol></li><li>文化与治疗<ol><li>从Transwell小下取出介质，加入700μl处理介质（工作介质添加了重组蛋白或药物抑制剂）的transwell小下。 </li><li>加入300μl的处理介质滴加于肠内的顶部，并允许它浸泡通过transwell小。重新定位的肠子是必要的。 </li><li>至少每日一次，或更频繁地更换处理介质取决于治疗试剂的半衰期。 </li></ol></li><li>治疗试剂的局部源制备蛋白浸泡琼脂糖珠<ol><li>悬浮琼脂糖珠在自己的存储容器，并转移100微升的琼脂糖珠到1.5 ml离心管中。 </li><li>填充管的其余容积用无菌1X博士osphate缓冲盐水（PBS）中并混合的珠子洗涤。将离心管中的齿条几分钟以使珠沉降至底部，然后从上珠粒顶端吸取掉PBS中。重新填充离心管中，用无菌1×PBS中，并重复洗涤过程两次以上。 </li><li>制备所关注的蛋白的两倍所需的治疗浓度。 </li><li>混合5微升的洗涤过的琼脂糖珠与5微升的2个蛋白存量，并轻轻搅拌中的蛋白质的珠。放置在冰上的管中1小时后，混合轻轻地每10-15分钟。 </li><li>把珠子上的肠子前在无菌1X PBS冲洗短暂的珠子。 </li></ol></li><li>琼脂糖珠安置<ol><li>轻轻将琼脂糖珠上使用吸管口用毛细管拉针肠子上。将珠子非常谨慎的粗糙处理会损害肠道，防止绒毛发展中的受伤部位。 </li><li>将两倍多珠需要进行分析，因为肠子会发生蠕动和大约一半的放置会滚下肠子在培养时间的珠子。 </li></ol></li><li>培养肠道内固定<ol><li>小心地从transwell小下面取出处理介质，并允许肠内空气干燥3分钟。 </li><li>加入1 ml固定液的transwell小室下2分钟，然后盖与2ml固定液为的固定时间剩余肠道的顶部。修正了4％多聚甲醛在o / n 4℃或2小时在室温。 </li></ol></li></ol> 2，修正了影像的肠子<ol><li> Wholemount成像<ol><li>将整个肠子（与内源性荧光）上用100微升1X DPBS的幻灯片。使用镊子轻轻地安排肠子。 </li><li>使用实验室的组织，以小心地取出DPBS，立即加入100微升70％的甘油，使组织MO再透明，提高成像的可能深度。 注:不是安装在70％甘油中的肠如果肠的进一步渗透是期望的，浸泡在几滴焦点清除溶液中的肠10-30分钟。吸管关闭对焦清晰的解决方案，并安装与安装清除肠道。 </li><li>每个四角盖玻片动用橡皮泥，拿起少量粘土使用的幻灯片和盖玻片之间的间隔，以保持盖玻片从压扁的肠道。 </li><li>密封盖玻片上施加，用棉签融化VALAP的幻灯片。 </li><li>图片上无论是直立或倒置的共聚焦显微镜的肠子。请参阅更详细的讨论。 </li></ol></li><li>固定肠子Vibratome切片（肠结构的剖视图） <ol><li>在7％琼脂糖嵌入肠子在小塑料模具（10×10×15毫米），并允许将琼脂糖块冷却和固化。 </li><li>从塑料模具和琼脂糖胶块与瞬间胶的vibratome收集阶段取出琼脂糖块。 </li><li>填用冰冷的1X DPBS收集阶段。 </li><li>切段在100-150微米之间的厚度。对于较厚的部分使用结算解决方案（在2.1.2节中描述），以可视化更深的横截面。 </li><li>倾从收集阶段vibratome节到6孔板中存储的阱，在4℃。 </li></ol></li><li>固定，vibratome切片组织免疫荧光染色<ol><li>将每口井5-12 vibratome节到24孔板与1X DPBS。 </li><li>删除1X DPBS和每孔加入500μl的通透的解决方案。轻轻摇动样品在室温下25分钟。 </li><li>透化后，用1×PBS洗涤样品3次。 </li><li>阻断样品中加入500μl封闭液中的至少30分钟。 </li><li>后堵，E交换网板用500μl稀释在封闭溶液中的初级抗体的阻断溶液并保持样品在4℃CO / N。 注:在冰冻和石蜡切片工作，以及一抗稀释液一般在vibratome部分工作得很好，但抗体需要核抗原修复一般不与本协议（ 例如，BrdU的）工作。 </li><li>翌日，每次用封闭溶液洗涤样品3次，持续15分钟。 </li><li>洗涤后，将500μl的稀释在封闭溶液中的二级抗体。包裹24孔板中的铝箔，以保护荧光团从光，并摇动样品45分钟至2小时，在室温。 </li><li>用1×PBS洗涤样品3次，每次15分钟，并在第2.4节中所述安装在滑轨的vibratome部分。 </li></ol></li><li>安装vibratome节<ol><li>准备幻灯片vibratome安装切割薄片双STICK胶带，并把它们沿着滑动的长边。 </li><li>小心地放置在幻灯片上的双面胶的5-10 vibratome节下降100μL1X PBS中。 </li><li>展开所有部分，并蔓延出来到对面的幻灯片单层。 </li><li>删除多余的琼脂糖或不均匀位，防止盖玻片从坐在平坦。 </li><li>小心使用实验室的组织来吸收多余的1X PBS来自各地的vibratome部分。 </li><li>加一滴含水安装介质上的vibratome部分的顶部，然后盖玻片。 </li><li>密封盖玻片上融化的VALAP应用，用棉签幻灯片。 </li><li>图片上无论是直立或倒置的共聚焦显微镜的vibratome部分。请参阅有关选择的成像系统的进一步的细节进行讨论。 </li></ol></li></ol> 3，现场全肠道的影像学肠子从fetuse收获ŠE12.5后，与所连接的大网膜原封不动，成功地利用上述系统开发绒毛文化。因此，本体外系统允许显影肠可重构的形态随时间的三维视图的活的z截面图像的捕捉。 <ol><li>建立实时成像<ol><li>使用瞬间胶，坚持个人的聚碳酸酯膜细网筛。这提供了一个支架支撑，以保持肠道到位直立共聚焦显微镜的镜片浸泡在下面。请参考讨论部分对选择实时成像显微镜进一步的细节。 </li><li>将网筛在中心以及文化板块。 </li><li>将解剖肠到聚碳酸酯膜，并在两端加一滴瞬间胶。仅使用足够的胶水贴上肠道，而不是大衣呢！ </li><li>添加酚红培养基免费聚碳酸酯以下膜在中心孔的培养板。 </li><li>加水至培养板的外边缘，以提供湿度。 </li><li>由于胎儿肠管发生在文化收缩蠕动，像波浪一样，加入100μl的1:在1X PBS溶液5甲苯噻嗪〜3毫升的媒体控制肠道而成像的运动。这个剂量控制肠道运动约8-10小时，不影响绒毛的发展。 </li><li>对于肠道的横向视图，嵌入在3-4％琼脂糖溶液活肠切成厚的部分（150-500微米）上vibratome。匹配与肠的刚度琼脂糖的刚性被切割和经验确定的发育​​阶段。一般情况下，3％的琼脂糖溶液可以很好地用于肠道年龄小于E14.5和4％的溶液可以很好地用于肠E15-E16。 </li><li>胶水vibratome部分以聚碳酸酯膜贴在网筛（如第3.1.1节），并培养第3.1.2-3.1.6所述。 </li><li>利用双光子共聚焦荧光显微镜（690-1,040纳米之间的激光激发）上直立架与可调表进行实时成像。 </li><li>双光子激光调谐到900nm的为用户提供了GFP信号的最佳分辨率深层渗透。它减少了损伤的组织，而成像。此外，激光的功率设定为尽可能低的排放量减少的组织损伤。通常，得到的GFP的良好激发，用12％的功率上的双光子激光。 </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

动态性和发展肠道复杂的组织相互作用的，需要三维可视化有这些形态发生事件的全面理解。随着不断变化的影像技术，能够检查绒毛形态进行了详细的发展/改善有了它，空间通信和交互的器官形成期的理解大大提高。 另一种方法培养整个肠子也进行了测试，但Transwell小系统仍是最可靠的更长的培养时间，并维持正常的集群和绒毛图案。三维培养系统（基质胶或胶原蛋白）?...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table><tbody><tr><td>Fine dissecting forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;11254-20</td><td>2 pairs</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>1x sterile Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Gibco&amp;nbsp;</td><td>14040-133</td><td>500 ml</td></tr><tr><td>6 well plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>24 well plates</td><td>Costar&amp;nbsp;</td><td>3524</td><td></td></tr><tr><td>60 x 15 mm petri dishes</td><td>Falcon</td><td>&amp;nbsp;451007</td><td></td></tr><tr><td>Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td><td>Corning Costar&amp;nbsp;</td><td>3428</td><td>6 inserts per plate</td></tr><tr><td>BGJb media</td><td>Invitrogen&amp;nbsp;</td><td>12591-038</td><td>500 ml</td></tr><tr><td>PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td><td>Gibco</td><td>&amp;nbsp;15140</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma&amp;nbsp;</td><td>A0278</td><td>make 5 mg/ml stock, filter, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td><td>Fisher</td><td>&amp;nbsp;21-180-10</td><td>remove the aspirator assembly and replace it with a 1,000 &amp;micro;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td></tr><tr><td>6 inch glass pasteur pipets</td><td></td><td></td><td></td></tr><tr><td>Capillary Tubes</td><td>World Precision Instruments&amp;nbsp;</td><td>TW100F-4</td><td>pull to needles</td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td>made in 1 x PBS, pH to 7.3</td></tr><tr><td>Culture plates</td><td>Falcon&amp;nbsp;</td><td>353037</td><td></td></tr><tr><td>Fine mesh stainless steel screen</td><td>purchase at hardware store</td><td></td><td></td></tr><tr><td>Polycarbonate membranes</td><td>Thomas scientific&amp;nbsp;</td><td>4663H25</td><td>alternatively, cut Corning Costar 3428 membranes off of transwell supports</td></tr><tr><td>Instant glue</td><td>purchase at hardware store</td><td>gel based preferrably</td><td></td></tr><tr><td>35 x 10 mm plates</td><td>Falcon&amp;nbsp;</td><td>351008</td><td></td></tr><tr><td>7% agarose</td><td>Sigma</td><td>&amp;nbsp;A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>minutien pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Phenol red free media (DMEM)</td><td>Gibco&amp;nbsp;</td><td>21063-029</td><td></td></tr><tr><td>Xylazine (100 mg/ml)</td><td>AnaSed&amp;nbsp;</td><td>139-236</td><td></td></tr><tr><td>Matrigel</td><td>BD</td><td>356231</td><td>basement membrane matrix, growth factor reduced, phenol red-free</td></tr><tr><td>3-4% agarose</td><td>Sigma&amp;nbsp;</td><td>A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>Imaging of fixed intestines</td><td></td><td></td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>vaseline</td><td>purchase at pharmacy</td><td></td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>lanolin</td><td>Sigma&amp;nbsp;</td><td>L7387</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>paraffin</td><td>Surgipath</td><td>39601006</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>70% glycerol in 1 x PBS</td><td></td><td></td><td></td></tr><tr><td>Focus clear and Mount Clear</td><td>CelExplorer Labs Co.</td><td>&amp;nbsp;F101-KIT</td><td></td></tr><tr><td>Modeling clay</td><td>purchase at art supply store</td><td></td><td></td></tr><tr><td>double stick tape</td><td></td><td></td><td></td></tr><tr><td>cotton applicator swabs</td><td></td><td></td><td></td></tr><tr><td>plastic molds, 10mm x 10mm x 5 mm)</td><td>Tissue Tek&amp;nbsp;</td><td>4565</td><td></td></tr><tr><td>slides</td><td></td><td></td><td></td></tr><tr><td>coverslips</td><td></td><td></td><td></td></tr><tr><td>lab wipe</td><td>Kimberly Clark</td><td>34155</td><td>lint free delicate task wipe</td></tr><tr><td>Theiler staging chart&amp;nbsp;</td><td></td><td></td><td>&lt;a href=&quot;http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf&quot;&gt;http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf&lt;/a&gt;</td></tr><tr><td>Leica SP5X confocal microscope&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Leica DMI 6000 stand&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Aqueous mounting medium (Prolong Gold)</td><td>Molecular Probes&amp;nbsp;</td><td>P36930</td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>24 well plate</td><td>Costar</td><td>&amp;nbsp;3524</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T-8787</td><td>used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td></tr><tr><td>Goat serum</td><td></td><td></td><td>used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td></tr><tr><td>Tween20&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;P9416</td><td></td></tr></tbody></table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

在胎儿小肠大部分形态过程已经推断出从固定的组织薄片，提供了发展阶段变化的快照。由薄的连续切片的三维信息可以是具有挑战性的，因为完美重构连续切片和保持组织在连续切片的适当定向的难度来解释。由格罗斯等人最近的研究结果，2011年重点在了解肠1的开发绒毛形态三维信息的重要性。单独使用标记的肠细胞的三维重建表明大多数肠上皮细胞的接触既心尖和基底表面。此外，肌动蛋白细胞骨架在上皮的顶端表面的三维重建表明肠腔是连续的，并且次要流明是第一个工件ectioning。这两点，伴随着interkinetic核移植的肠上皮示范，明确了发展中肠上皮为假复层上皮，不分层，如以前认为1。观察到上皮三维的能力是开创性到证明了这一点，重新定义上皮形态在胎儿肠。用多光子成像技术和三维重建软件的演化，能够可视化完好，显影机构正在迅速提高。双光子激发可损害较小穿透更深的组织具有较高的分辨率。双光子成像和三维重建整个胎鼠肠中的沃顿等人 ，2012帮助定义绒毛生长2的格局。在这里，我们描述了整个器官培养系统，使离体的发展是文化系统的绒毛和扩展，使肠道是三维的开发过程中进行成像。

胎儿肠的整个外植体的培养可为位置的信号分子的坐标肠的发展，分配，和持续时间的分析，因为该系统能够与药理学试剂或重组蛋白质的信令的操作。 Transwell小培养系统（ 图1A中 ，从沃尔顿等再现。，2012）2提供了一种空气-液体界面，这使得它能够与毒品或蛋白质浸泡琼脂糖珠放置到组织中，从而建立本地信令资源（ 图1B ）。肠子从胚胎E10到E18....

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

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.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

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

14.8K 次观看.  University of Michigan. 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. 

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

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

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

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

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

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

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

科研

发育生物学

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

生物工程

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

癌症研究

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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

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

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

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

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

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

免疫与感染

A System for ex vivo Culturing of Embryonic Pancreas

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

A System for ex vivo Culturing of Embryonic Pancreas

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

一个系统<em&gt;体外</em培养的胚胎胰腺

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

生物学

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

大鼠肠隐窝分离和肠道类器官生成

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

作者聚焦:大鼠肠道类器官的产生和操作  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

老鼠胎儿整肠文化系统

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活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

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小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

大鼠肠道类器官的产生和操作