Name: effective isolation of functional islets from neonatal mouse pancreas
Uploaded: 2017-01-06T11:30:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

动物的使用遵循协议中号规定的程序/一百八十一分之十一由范德比尔特机构动物护理和使用委员会批准谷。 CD1或CBA / BL6小鼠从商业供应商处购买和交叉在范德比尔特动物设施获得新生小鼠。 1.小鼠制备，股票解决方案和设备<ol><li>对于鼠标交叉:成立一个跨鼠标并记录日期堵漏帮助实验计划。 CD1小鼠通常交配后给周围19天诞生。使用CD1或CBA / BL6穿越获得CD1或CBA / BL6新生儿，分别。两条线之间Intercrosses利用CD1雌性和CBA / BL6男性。 </li><li>对于胶原酶股票:体重200毫克IV型胶原酶具有高精确度的平衡。转移至50毫升离心管中。溶解在40ml的HBSS用的 Ca 2+ / Mg 2+离子 （1.26和0.5mM，分别地）做出5毫克/毫升贮备液。 <ol><li>允许胶原酶溶解于轻轻摇动约30分钟。分装并保存在-20℃的股票，这对于至少一年保持有效冻结的解决方案。 </li></ol></li><li>高压灭菌消毒几双镊子，剪刀和P20技巧解剖和胰岛采摘。 </li><li>对于1M的葡萄糖股票:称取9克葡萄糖50毫升离心管中，加入50毫升RPMI 1640媒体溶解葡萄糖。保持在4℃直至使用，但不超过6个月。 </li></ol> 2.工作方案注:胰岛分离的日子，准备以下试剂。 <ol><li>对于IV型胶原酶工作液:解冻，并稀释冰上胶原酶股票。免除0.15毫升股票进入各1.7毫升离心管。添加1.35毫升HBSS与钙 /镁离子 。倒置该管5次。留在冰上15分钟。 <ol><li>旋转以高速在微量3分钟。转移上清液至新管。留在冰上，直到使用。丢弃不溶性碎片管。 </li></ol></li><li>对于完全RPMI 1640培养基:添加2.5毫升的1摩尔葡萄糖，50毫升热灭活的FBS和5mL的100×青霉素 - 链霉素库存入500毫升的RPMI 1640培养基。留在冰上，直到使用。 </li></ol> 3.胰腺分离和消化<ol><li>安乐死用异氟烷新生儿，根据批准的方案随后断头。 </li><li>搁浅了，肚子朝上鼠标。用70％乙醇消毒喷雾。 </li><li>提起腹部皮肤与镊子和纵向切开皮肤和肌肉层以及一对剪刀的中线，从生殖器区域到肋笼。这将打开腹腔暴露所有的内脏。 </li><li>传输所有内脏100 cm的天线用一对镊子。定位胰腺，这是一个分散的器官与粘附到胃，脾和粘附到十二指肠11腹侧部的背部。 </LI&gt; <li>添加HBSS入菜淹没的器官。在溶液中，胰腺组织不再执着于十二指肠，胃或脾。这是因为其典型的白色下解剖范围容易识别。使用一对镊子的从周围组织和传送远离剥离胰腺组织到一个新的60mm培养皿用HBSS。 </li><li>切用剪刀每个胰腺成片比在任何尺寸为5mm小。转移到1.5毫升离心管中。多达五个胰腺（年龄小于P7）可以在一个管被消化。对于P8-P16的小鼠，使用每个胰腺一管。 </li><li>加0.5 - 1毫升胶原酶工作液到每个管（0.2毫升每个P1-P7胰腺使用0.5mL的每个P8-P16胰腺）。留在37℃培养箱管长达15分钟。反转管每分钟两次。 </li><li> 〜5分钟后，点击管监视胰腺组织的消化状态。继续消化过程，直到大部分胰腺组织表现为零散的细胞团，直径&lt;2毫米。裂解液出现浑浊，这是由于正常的腺泡细胞自溶。 </li></ol> 4.裂解液洗<ol><li>在微型旋转溶解物在500×g离心10秒。用P1,000的Pipetman去除上清层。上清应该出现浑浊，但缺乏细胞团。不使用抽吸，其可以迅速地吸走在底部的组织碎片由于一些粘DNA的存在。 </li><li>加入1毫升RPMI 1640完整的媒体。轻轻拍打管悬浮分散的裂解液。反转向上和向下10倍。重复步骤4.1。 </li><li>重复步骤4.2两次或者直到上清出现明显的。在1毫升完全RPMI 1640培养基重悬水洗胰腺片段。 </li></ol> 5.胰岛分离注:对于胰腺小的数字，直接用手采摘如下（5.1），可用于胰岛ISOL通货膨胀。对于大量的胰腺（&gt; 6），在5.2中列出的方法是首选。 <ol><li>直接手工采摘: <ol><li>从步骤4.3传输裂解液60毫米的菜。加入5毫升完全RPMI 1640媒体。在解剖显微镜下把。 </li><li>建立在显微镜明视场照明。调整光强度到最大功率的〜50％。在此条件下，胰岛显示为淡粉红色簇，而腺泡细胞为暗不规则形群集（ 图1A）。 〜50X（结合目标的能力和目镜）的放大倍数，建议提供可视化胰岛的最佳方法，同时将枪头开放采摘。 </li><li>拿起胰岛与P20的枪头，同时避免腺泡集群。分配胰岛富集馏分到一个新的60mm培养皿的RPMI完全培养基（ 图1B）。 </li><li>重复手工采摘过程中的两倍多。离开纯胰岛在新60毫米培养皿用4mL的完全RPMI培养基（ 图1C）。 </li></ol></li><li>梯度离心法: <ol><li>制备是在10.77克/毫升的密度预装用2mL多聚蔗糖和钠泛影15毫升离心管中。离开冰管，直到使用。 </li><li>传送来自步骤4.3的悬浮液到用巴氏吸管的多聚蔗糖和钠泛影葡胺溶液的顶部。请勿打扰底层。从多达10个新生或2 P17胰腺组织可以加载到每15毫升管中。 </li><li>旋在〜600 XG所述管中的摆动转子15分钟，在4℃。使用制动开关设定。 </li><li>离心后，转移媒体和相间层（请注意，理想的操作条件下，胰岛将位于离心后的多聚蔗糖和培养基之间的相间）到一个新的15毫升管中。加入10 mL媒体，轻轻但调匀 。 </li><li>降速胰岛浓缩部分在300×g离心5分钟。取出洗净媒体。重复洗一次完全RPMI媒体。悬浮颗粒在4毫升媒体，它主要含有胰岛和导管（ 图1D）。 </li><li>手工挑选胰岛如第5.1节所述。 </li></ol></li></ol> 6.化验GSIS在分离的胰岛<ol><li>孵育从步骤5.1.4在完全RPMI培养基的胰岛在37℃组织培养孵化2小时。 </li><li>使用移液管除去在解剖显微镜下的RPMI。添加3与2.8毫米的葡萄糖毫升KRB溶液到板3。摇动10次洗小岛。删除KRB解决方案。 <ol><li>重复洗涤过程中一次。孵育用3mL KRB溶液胰岛在37℃在培养箱中1小时。 </li></ol></li><li>等分试样1毫升KRB到12孔板的每个孔中。预暖KRB的股票和板块37℃的孵化器。 </li><li>与预热KRB洗两次胰岛按照6.2。横贯FER 10胰岛到预热板的各孔中。孵育在37℃培养箱。 </li><li> 45分钟后，撤上清50μL的。这包含在基础葡萄糖分泌胰岛素溶液。 </li><li>加入32微升500毫米的葡萄糖。摇动板20倍到该溶液中混合。孵育在37℃培养箱。摇动板5次每隔5分钟。 </li><li> 45分钟后，撤50微升上清液。这是高糖分泌胰岛素。 </li><li>传输所有胰岛1.5mL管。冻结在-20℃30分钟。解冻在室温下5分钟。重复冷冻和解冻过程。 </li><li>添加0.5mL的70％乙醇的20mM HCl中。留下过夜。这是留在小岛的胰岛素。 </li><li>用常规ELISA法测定胰岛素水平，先前3所述。 </li></ol>

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The authors declare no competing financial interests for the described work.

在这里，我们提供胰岛分离的一步一步的协议，从是传统灌注太小胰腺。据预计产量胰岛准备好所有基于胰岛研究，如β细胞的纯化，基因表达分析，胰岛β细胞的成熟，增殖，细胞应激反应，细胞存活，代谢和功能GSIS维护等 。这将是，以我们所知，第一次详细目视协议，引导新的研究人员从新生小鼠进行胰岛分离的。如果没有这些视觉助手，广泛的试验和错误是预期的大多数研究人员实?...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table><tbody><tr><td>Collagenase Type IV</td><td>Sigma Aldrich, St Louis, MO</td><td>C5138</td><td></td></tr><tr><td>1x HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech/Cellgro, Manassas, VA</td><td>MT21020CV</td><td>If HBSS wihout Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; is obtained, CaCl&lt;sub&gt;2&lt;/sub&gt; and MgSO&lt;sub&gt;4&lt;/sub&gt; can be added to 1.26 and 0.5 mM, respectively.&amp;nbsp;</td></tr><tr><td>RPMI 1640 w/o glucose</td><td>Thermo Fisher Scientific/Life Technologies, Waltham, MA</td><td>11879-020</td><td>Glucose needs to be added to specific levels to not interfere with subsequent islet usage.&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma Aldrich, St Louis, MO</td><td>G-7021&amp;nbsp;</td><td></td></tr><tr><td>polysucrose and sodium diatrizoate solution</td><td>Sigma Aldrich, St Louis, MO</td><td>Histopaque-10771</td><td></td></tr><tr><td>Stereoscope</td><td>Carl-Zeiss, Oberkochen, Germany</td><td>Stemi2000</td><td></td></tr><tr><td>Stereoscope</td><td>Leica, Wetzlar, Germany</td><td>Leica M165</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5417C</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>15-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-666</td><td></td></tr><tr><td>50-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-658</td><td></td></tr><tr><td>Precision balance</td><td>VWR, Radnor, PA</td><td>VWR-225AC</td><td></td></tr><tr><td>Microfuge tubes</td><td>VWR, Radnor, PA</td><td>87003-294</td><td></td></tr><tr><td>Pipetman P1000</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123602</td><td></td></tr><tr><td>Pipetman P20</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123600</td><td></td></tr><tr><td>100 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-088</td><td></td></tr><tr><td>60 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-168</td><td></td></tr><tr><td>12-well plates</td><td>VWR, Radnor, PA</td><td>665-180</td><td></td></tr><tr><td>Scissor</td><td>Fine Scientific Tools, Foster City, CA</td><td>14080-11</td><td></td></tr><tr><td>Tweezers</td><td>Fine Scientific Tools, Foster City, CA</td><td>5708-5</td><td></td></tr><tr><td>CD1 mice</td><td>Charles River Laboratories, Wilminton, MA&amp;nbsp;</td><td>CD-1</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>C57BL/6J</td><td></td></tr><tr><td>B6CBAF1/J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>B6CBAF1/J</td><td></td></tr></tbody></table>

effective isolation of functional islets from neonatal mouse pancreas

从大型哺乳动物的胰腺基于灌注胰岛隔离协议确立。这种协议是很容易地在许多实验室由于大尺寸的胰管，允许准备注射胶原酶和随后的组织灌注的进行。与此相反，从小型胰腺胰岛分离，像新生小鼠，是具有挑战性，因为灌注是不容易在小胰腺实现的。在这里，我们描述了从恢复视觉协助新生小鼠胰岛相当数量的详细的简单过程。新鲜解剖整个胰腺用在37℃下溶解在Hanks&#39;平衡盐溶液（HBSS）0.5毫克/毫升胶原酶IV消化，在微量离心管。管定期窃听，以帮助组织扩散。当大部分组织的分散，以小簇约1毫米，裂解物，用10％胎牛血清（FBS）洗涤三到四倍用培养基。胰岛集群，缺乏可识别的腺泡组织，然后可以根据解剖立体镜来回收。这种方法恢复20 - 80小到每个刚出生的小鼠胰腺大型小岛。这些胰岛适合大多数可想到下游测定法，包括胰岛素分泌，基因表达，和培养。呈现胰岛素分泌测定法的实例，以验证隔离过程。遗传背景和消化的程度是决定产量的最大因素。高活性新鲜的胶原酶溶液是首选，因为它在内分泌外分泌隔离艾滋病。阳离子在洗涤所有的解决方案和胎牛血清存在下[钙（Ca 2+）和镁（Mg 2+）] /拾取介质所必需的具有适当的完整性胰岛的良好的产率。具有良好的对比度和放大倍率解剖范围也将有所帮助。

从每个小老鼠胰腺胰岛80 - 在最佳条件下，该方法可以产生20。这个数量取决于遗传背景，年龄的小鼠，并加以回收胰岛的大小。这些常用的，CD1出孕育和C57BL / 6J纯种小鼠产生比CD1和C57BL / 6或商业B6CBAF1之间的杂交尺寸更小更少的胰岛/ J小鼠做的。直接用手采摘一般了数量较少的小岛，可能是由于小岛排除这可能是很难当外分泌组织（ 图1A-C）混合的认可。梯?...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

我们在此描述了一个协议，从新生小鼠分离胰岛完好无损。胰腺被部分用胶原酶消化，接着洗涤和手采摘。 20 - 80胰岛集群可以每胰腺从新生小鼠，这是适合于几个胰岛研究而获得。

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  Vanderbilt University Medical School. 我们在此描述了一个协议，从新生小鼠分离胰岛完好无损。胰腺被部分用胶原酶消化，接着洗涤和手采摘。 20 - 80胰岛集群可以每胰腺从新生小鼠，这是适合于几个胰岛研究而获得。 

视频: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

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

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

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

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

免疫与感染

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

生物学

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 ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

新生小鼠胰腺胰岛功能的有效隔离

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