作者感谢Guanhao Biotech的Wei Zhang提供脱细胞支架。感谢彭鹏鹏有意思的讨论。这项研究得到了国家自然科学基金资助（项目编号:313122021）的资助。

所有实验均获得北京大学机构动物保护与使用委员会（IACUC，IACUC no.COE-Luo-1）的批准。 岛屿隔离<ol><li> 试剂和设备的制备。 <ol><li>在HBSS中重建胶原酶P粉末（2U / mg）以制备5mg / mL溶液，并通过0.22μm过滤器过滤以除去细菌。在15 mL锥形管中制备0.6 mL胶原酶P等分试样溶液，并保存在-20°C。 注意:在使用过程中，每个等分试样用HBSS稀释，得到最终浓度为0.5 mg / mL或1 U / mL的6 mL工作溶液（足以治疗3只小鼠）。工作溶液保存在冰上，即可在1小时内立即使用。不能恢复或重新冻结工作解决方案以便额外使用。 </li><li>通过在HBSS中加入FBS（2.5％）和P / S（1％）来制备中和溶液;将溶液保持在冰上。准备60 mL中和溶液至中t 6小鼠。 </li><li>对于胰岛培养基，向RPMI 1640培养基中加入D-葡萄糖（7mM），FBS（10％）和P / S（1％）。 </li><li>将手术器械在115°C高压灭菌30分钟和15 psi的压力。 </li></ol></li><li> 胰腺通货膨胀。 <ol><li>为6只小鼠（12周龄）准备12mL胶原酶工作溶液，如步骤1.1.1所述。向10mL注射器中加入9 mL工作溶液，并将注射器连接到27 G静脉注射针上。将注射器存放在冰上，并在1小时内使用溶液。 </li><li>通过颈椎脱位安乐死小鼠。将鼠标放在纸巾上的仰卧位置，尾巴指向操作者。用70％乙醇喷雾全身，使其完全湿润。 </li><li>做一个V型切口，从生殖器区域开始并延伸到隔膜，使用解剖剪刀和镊子。将皮肤折叠在胸部以完全露出腹腔。 </li><li>移动肠对右侧的小鼠，并暴露胰腺和胆总管。用镊子仔细抓住十二指肠，并拉扯直到胆管拉紧（ 图1A -1和A-2 ）。 </li><li>找到通往肝脏的门静脉和胆管。用微观止血钳夹住门静脉和胆管。 注意:夹持防止过多的出血和胶原酶进入肝脏。 </li><li>在用镊子保持肠道的同时，找到连接胆管和十二指肠的壶腹的位置。通过壶腹将27G静脉注射针插入胆总管（ 图1B -1和B-2 ）。 </li><li>分配约200μL胶原酶工作液，检查插管是否全部通过胆管。如果胶原酶溶液开始填充管道（ <s图1C- 1和C2 ），针位于导管的内腔中;使用动脉止血钳夹住包含针头的管段，并在1分钟内以缓慢且恒定的速率分配2 mL溶液的其余部分（ 图2A -1 ， A-2和B-1 ）。 注意:如果管道周围的组织开始膨胀（ 图2B -2 ），针头已经穿过胆管壁，需要重新定位针头并再次插入胆管。类似地，如果十二指肠开始膨胀（ 图2C -1和C-2 ），则需要调整动脉止血钳，并重新插管胆管。由于胶原酶溶液填满胰腺，十二指肠附近的组织首先膨胀，其次是区域n靠近胰尾。胰尾的灌注（脾叶）对于最大化胰岛产量是重要的。 </li><li>胰腺完全充气后（ 图2C -1 ），将肠推至老鼠的左侧，并从降结肠开始移除胰腺。使用镊子提起肠，并用另一对镊子将其与胰腺分开。继续去除胰腺，直到其从胃的顶部未附着。最后，将胰腺从腹部取出，并将其从剩余的脾脏中切除。 注意:整个分离应该快速进行，因为在去除过程中消化继续进行。 </li><li>将胰腺放在空的15-mL锥形管中，放在冰上。对剩余的小鼠重复上述步骤。所有胰腺应在1小时内进一步处理，并迅速按照步骤1.3，以防止胶原酶P过度消化/ LI&gt; </ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/54995/54995fig1.jpg" /> 图1: 显示胆管插管和用胶原酶溶液灌注胰腺的照片。 （ A1 ）拉出十二指肠直至胆管紧张。 （壶腹:十二指肠表面上的三角形，乳白色区域;胆管:表面上的线状乳状结构）。 （ B1 ）从壶腹将针插入胆管。 （ C1 ）用注射酶使胰腺充气。 （ A2，B2和C2 ）分别在A1，B1和C1中显示的程序的卡通图像。 <a href="//ecsource.jove.com/files/ftp_upload/54995/54995fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2: 插管故障排除。 （ A1 ）针尖插入胆管内腔。 （ A2 ）管道充满酶溶液。 （ B1 ）将针插入胆管内腔，并注入蓝色染料。 （ B2 ）由于不适当的插管，针在胆管下方，并且在分配蓝色染料后仅观察到充气胶囊。 （ C1 ）胰腺扩张证明了成功的插管。 （ C2 ）由于不适当的夹持，蓝色染料进入十二指肠并引起膨胀。 <a href="//ecsource.jove.com/files/ftp_upload/54995/54995fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="3"><li> &lt;强&gt;胰岛的消化和净化。 <ol><li>将含有灌注胰腺的锥形管在37℃下孵育17分钟。 注意:孵育时间可能随着动物的年龄和种类而变化。 </li><li>通过加入7mL中和溶液终止消化，并将管置于冰上。 </li><li>通过剧烈摇动管（ 例如， 10秒钟20次）使组织分离，直到获得细小的组织颗粒。 注意:如果胰腺完全解离，胰岛产量将会很低。 </li><li>通过0.5 mm丝网过滤消化的组织样本，以去除任何未消化的组织块。将胰岛悬浮液收集在新的50mL锥形管中。 </li><li>在230×g和4℃下将管离心3分钟，并小心地倒出上清液，而不会干扰组织团粒。 </li><li>通过轻轻旋转或p旋转，将来自3只小鼠的沉淀物重悬于4mL聚蔗糖密度梯度中暂停上下几次。慢慢地将4mL HBSS从管的侧面移至聚蔗糖溶液的顶部。 注意:两个解决方案应该是具有清晰界面的良好分离的层。 注意:HBSS的添加应小心进行，而不会影响底部的聚蔗糖密度梯度。 </li><li>通过选择非常慢的加速速率，将悬浮液在900 xg和4℃下离心20分钟。无刹车结束离心。 注意:这是从外分泌细胞中纯化胰岛的步骤，其中大多数胰岛迁移到聚蔗糖层和HBSS层之间的界面以及大多数外分泌细胞沉降在底部。 </li><li>使用大口径15 mL移液管去除8 mL上清液的全部。将溶液通过倒置的70-μm过滤器。 注意:胰岛将进一步纯化并保留过滤器，而外分泌细胞将通过过滤器。 注意:聚糖密度梯度为细胞毒性;过滤将帮助小岛摆脱聚蔗糖。 </li><li>将2 mL冷中和溶液吸入35 mm培养皿中。将细胞过滤器右侧反转，将保留胰岛的表面浸入溶液中，轻轻摇动释放胰岛。 </li><li>在显微镜下使用带有白色尖端的20-μL移液管手工挑选胰岛。 注意:胰岛是淡黄色，紧密的细胞聚集体（ 图3A ），而在解剖显微镜下，污染的外分泌组织或细胞具有黑色和松散的结构。 </li><li>将大约200个胰岛放在35毫升培养皿的培养皿中，并在37℃下，在5％CO 2培养箱中培养12小时。 注意:通常可以从一个12周龄的C57BL / 6J小鼠收获150-300只胰岛。在培养过程中，胰岛素易形成聚集体，大胰岛（&gt;300μm）易发生中心坏死（ 图1B ，插图）。将悬浮液充分摇匀，将胰岛均匀分布在盘中并减少结块。 </li></ol></li></ol> 2.脚手架上的胰岛文化注意:DCS具有约79％的孔隙率，约0.6mm的厚度和12-300μm的孔径。 <ol><li>将支架切成7 mm圆盘，将其浸泡在70％乙醇中，并用HBSS洗涤。将支架置于24孔组织培养插入物中。 注意:过夜培养后，当新鲜胰岛恢复时，胰岛呈现明亮而紧密，边界光滑（ 图3B ）。 </li><li>旋转菜肴，并使用20微升移液管将白菜从中心收集胰岛。使用移液管将胰岛转移到支架（250个胰岛/支架），并加入2mL培养基那个井在移植前培养胰岛12小时。 </li></ol> 3.在EFP网站移植胰岛<ol><li> 受体小鼠糖尿病诱导。 <ol><li>将C57BL / 6J小鼠（超过10周龄）放在带有供水但没有食物的新鲜笼子中。在诱导糖尿病前，将小鼠快速放置10小时。 </li><li>通过将0.1M柠檬酸与0.1M柠檬酸钠混合，制备pH值为4.2-4.5的缓冲溶液。在新鲜制备的缓冲液中溶解STZ（10mg / mL），并将溶液通过0.22μm过滤器进行灭菌。 注意:STZ对光敏感，10分钟内失去活性;始终在注射前准备新鲜的STZ溶液。 </li><li>每只C57BL / 6J小鼠以140-150mg / kg的剂量向STZ腹膜内注射每只小鼠。 注意:剂量取决于动物的年龄和种类。建议进行小规模的剂量优化对于给定的物种，在开始正式实验之前。 </li><li>收集尾静脉血，并在STZ注射后第2,3和4天用葡萄糖计监测血糖。 注意:当动物连续两天高血糖（非空腹血糖&gt; 16.7 mM）时，准备进行胰岛移植。 </li></ol></li><li> 将胰岛移植到EFP网站。 <ol><li>用戊巴比妥麻醉麻醉腹膜内给药（50mg / kg）。将鼠标放在纸巾上的仰卧位置，尾巴指向操作者。大量剃除腹部以从切口部位周围去除毛皮。将四肢贴下来，用交替的酒精完全擦拭皮肤，碘循环湿巾以圆形方式移动，对切口部位进行消毒。用无菌悬垂将鼠标悬垂，只允许进入切口区域。通过腹膜壁进行7毫米切口中线，靠近生殖器区。 注意:手术中使用的所有仪器（包括手套）均应无菌。 </li><li>使用镊子轻轻抓住并从腹腔中取出EFP。将EFP涂抹在湿润的无菌纱布上。将含有胰岛的支架放在EFP上，并折叠EFP以包装移植物。通过缝合EFP与可吸收的6-0缝线来确保胰岛和EFP之间的直接接触。使用浸泡的棉签，用无菌盐水湿润EFP的表面，以防止EFP变干。 </li><li>将EFP轻轻放回腹腔。通过缝合腹膜壁并用伤口夹夹住真皮层，关闭切口。 </li><li>皮下注射丁丙诺啡（0.1 mg / kg）作为镇痛药。 </li><li>皮下注射1 mL盐水以防止脱水。 </li><li>将鼠标放在加热垫上的笼子中，直到鼠标从麻醉恢复。 </li><li>检查非禁食葡萄糖两天后，从尾静脉收集血液。如果检索移植物，重复上述移植手术，轻轻拉出EFP，使用3-0缝线连接邻近附睾的脂肪垫的末端，封闭血管，外植EFP移植物，并保存用于组织学。 注意:推荐胰岛应在3天内再次使受体小鼠高血糖，从而确认移植物的功能。 </li></ol></li></ol>

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作者没有什么可以披露的。

胰腺灌注和消化时间是影响胰岛产量和质量的两个关键参数。 Moskalewski首先报道了使用粗胶原酶混合物来消化切碎的豚鼠胰腺11 。 Lacy 等人报道将酶注入管道系统灌注胰腺，大大提高了胰岛产量12 。酶的导管灌注允许胰腺表面积最大程度地暴露于酶，与消化胰脏13相比，导致更均匀的消化和更完整的胰岛的释放。根据我们的经验，胆管?...

1型糖尿病（T1D）是一种自身免疫性内分泌疾病，其中胰岛细胞被免疫系统消融，使患者依赖注射外源性胰岛素的整个生命。埃德蒙顿方案代表胰岛移植临床研究的里程碑;胰岛通过门静脉灌注并移植到肝内部位1 。然而，两个主要障碍 - 供体胰岛来源不足和胰岛移植不良 - 阻止胰岛移植的广泛成功2 。通常，需要从三个尸体捐赠者收集胰岛以扭转一名患者的高血糖状况;这是由于胰岛分离程序的产量低，移植后的胰岛损失。特别是，虽然移植后的小岛沐浴在富氧血液中，但是与血液的直接接触往往会诱发即时血液介导的炎症可能导致胰岛急性损失的原始反应（IBMIR）。长期来看，患者胰岛素逐渐消失占临床组患者糖尿病逆转率的下降，第一年可能达到90％，下降到30％，10％降至2和5移植后几年，分别为3 。 肝外部位的胰岛移植一直是减少胰岛与血液直接接触的有吸引力的策略，同时将肝移植置于与肝内输注相比更明确的位置。有研究肾囊中，眼睛，肌肉，脂肪垫和皮下空间在过去几年中进行的，这表明在这些网站胰岛能够生存和功能恢复正常血糖4。此外，这些部位的胰岛是可回收的，使得可以进行活组织检查或甚至进一步的替换手术。肝外因此它表明临床移植的巨大潜力5 。 基于生物材料的支架已经被深入研究用于细胞移植和组织工程。三维（3D）支架通常含有多孔结构，可用作细胞模板，以产生细胞的空间结构/组织或作为储存器提供生物活性线索的控制释放。支架也由聚（乙交酯-L-丙交酯） 6 ，聚（二甲基硅氧烷） 7和热塑性聚（氨基甲酸酯） 8等聚合材料制成，用于EFP中的移植胰岛。相比胰岛的移植直接，利用支架的发现通过防止胰岛的泄漏到腹腔9,10减少胰岛损失，从而提供机械保护和MODU延缓局部炎症反应。因此，可以开发支架以促进在移植部位7的移植。 在本研究中，我们打算在EFP中展示胰岛移植的范例，在使用DCS的小鼠模型中进行。由于与合成产品相比，优于生物相容性和更多的天然多孔结构，近年来由细胞外基质衍生的支架引起了极大兴趣。在这里，我们描述了从C57BL / 6J小鼠以高产量获得胰岛的强力分离方案。然后用牛心包膜处理的DCS用胰岛接种，将移植物移植到同源性糖尿病模型中的EFP。在10天内实现小鼠中的正常血糖，并保持长达100天，直到移植物移除。

<table><tbody><tr><td>Dissecting scissor</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Forceps</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>0.5 mm diameter wire mesh</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Artery hemostatic clamp</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Microscopic hemostatic clamp</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Hemostatic forceps</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Absorbable 6-0 PGLA sutures&amp;nbsp;</td><td>JINHUAN</td><td></td><td>With needle</td></tr><tr><td>Wound clip</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Gauze</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Sterile drapes</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>10mL syringe</td><td>JINGHUAN</td><td></td><td></td></tr><tr><td>1 mL syringe</td><td>JINGHUAN</td><td></td><td></td></tr><tr><td>27G intravenous needle</td><td>JINGHUAN</td><td>0.45x15 RWSB</td><td></td></tr><tr><td>1.5 mL Eppendorf tube</td><td>Axygen</td><td></td><td></td></tr><tr><td>15mL conical tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50mL conical tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>35mm Non-treated&amp;nbsp; Peri-dishes</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3422</td><td></td></tr><tr><td>0.22 &amp;mu;m filter</td><td>Pall</td><td>PN4612</td><td></td></tr><tr><td>10 mL serological pipet</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Pipet filler S1</td><td>Thermo Scientific</td><td>9501</td><td></td></tr><tr><td>Pipette (2-20&amp;mu;L)</td><td>Axygen</td><td>AP-20</td><td>AXYPET&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Gibco</td><td>C14175500CP</td><td></td></tr><tr><td>Collagenase P</td><td>Roche</td><td>COLLP-RO</td><td></td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11879-20</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>D-glucose</td><td>Gibco</td><td>A24940-01</td><td></td></tr><tr><td>Glucose meter</td><td>Roche</td><td></td><td>ACCU-CHEK</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Streptozotocin</td><td>Sigma</td><td>V900890</td><td>Vetec&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>Chloral hydrate</td><td>J&amp;amp;K</td><td>C0073</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>71497</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C2404</td><td></td></tr><tr><td>Iodophors</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>C57BL/6J, 10-12 weeks old</td><td>VitalRiver</td><td></td><td>Beijing, China</td></tr><tr><td>Decellularized scaffold</td><td>Guanhao Biotec</td><td>131102</td><td>Guangzhou, China</td></tr></tbody></table>

scaffold-supported transplantation of islets in the epididymal fat pad of diabetic mice

胰岛移植已被临床证明在治疗1型糖尿病方面是有效的。然而，目前的肝内移植策略可能引起急性全血反应，导致胰岛移植不良。在这里，我们报告了在肝外移植部位胰岛移植的强效方案 - 附睾脂肪垫（EFP） - 在糖尿病小鼠模型中。描述了分离和纯化来自C57BL / 6J小鼠的胰岛的方案，以及通过将胰岛接种到去细胞化支架（DCS）上并将其植入到同源基因C57BL / 6J小鼠中的EFP位点上进行的移植方法，所述小鼠呈现为糖尿病通过链脲霉素。含有500个胰岛的DCS移植物在10天内扭转了高血糖状态，而没有DCS的游离胰岛素需要至少30天。正常血糖维持长达3个月，直到移植物被移除。总之，DCS加强了胰岛的植入他的EFP肝外部位，可以很容易地被检索，并且可能为调查支架材料提供可重复和有用的平台，以及成功进行胰岛移植所需的其他移植参数。

我们使用微观止血钳进行的夹紧方法与缝合结扎技术相比是直接且节省时间的。大约需要4小时来分离和纯化来自6只小鼠的约1,200只胰岛。新鲜分离的胰岛在光学显微镜下通常具有粗糙的周边（ 图3A ）。一旦胰岛从隔离过程中恢复，它们看起来明亮而紧密，并获得光滑的表面。然而，紧张的隔离仍然可以诱导细胞死亡，导致细胞从胰岛表面的脱落，?...

Watch this Scientific Journal Video about Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice at JoVE.com

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

该方案证明了鼠胰岛分离和接种到脱细胞支架上。将脚手架支持的胰岛移植到链脲霉素（STZ）诱导的糖尿病小鼠的附睾脂肪垫中。胰岛在移植部位存活并逆转高血糖状况。

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 ...

scaffold-supported-transplantation-islets-epididymal-fat-pad-diabetic

Research

JoVE Journal

Bioengineering

10.0K 次观看.  Peking University. 该方案证明了鼠胰岛分离和接种到脱细胞支架上。将脚手架支持的胰岛移植到链脲霉素（STZ）诱导的糖尿病小鼠的附睾脂肪垫中。胰岛在移植部位存活并逆转高血糖状况。 

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

补肾胶囊对糖尿病小鼠的胰岛移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

生物学

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

生物工程

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING)

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG hOMING

Here, we present a protocol for in vivo validation of hydrogel-based cell therapy, illustrated by the example of islet transplantation. h-Omental Matrix Islet filliNG (hOMING) implantation allows implantation of a cell-hydrogel mixture between the omental layers, near to blood vessels, to maximize engraftment in a proper metabolic environment.

利用 h-oig境外矩阵子 filling (homing) 进行室内胰岛移植

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

使用附睾白色脂肪组织进行脂肪覆盖的胰岛移植

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

尽量减少小鼠肝内胰岛移植期间输注后门静脉出血

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

小鼠骨髓胰岛移植的一种方法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Murine Model of Islet Transplantation: A Surgical Method to Infuse Pancreatic Islets in Portal Vein and Prevention of Postoperative Bleeding

Source: Gou, W. et al. <a href="https://www.jove.com/v/62530">Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.</a> J. Vis. Exp. (2021)
In this video, we describe a procedure to infuse pancreatic islet cells into the&#160;hepatic portal vein, followed by application of gel foam to successfully prevent bleeding. Postoperative bleeding is a common cause of high mortality during islet transplantation.

胰岛移植小鼠模型:一种在门静脉输注胰岛的手术方法及预防术后出血

Source: Gou, W. et al. Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice. J. Vis. Exp. (2021)
In this video, ...