The authors would like to thank Bethany Carboneau (Vanderbilt University) for critical reading of this manuscript. We also thank Anastasia Coldren (Vanderbilt University Medical Center Islet Procurement and Analysis Core) for islet isolations, and Dr. Alvin C. Powers (Vanderbilt University Medical Center) and Dr. David Jacobson (Vanderbilt University) for use of their centrifuge and tissue culture facility. This research involved use of the Islet Procurement and Analysis Core of the Vanderbilt Diabetes Research and Training Center supported by NIH grant DK20593. This work was supported by an American Heart Association Postdoctoral Fellowship (14POST20380262) to R.C.P., and grants from the Juvenile Diabetes Research Foundation (1-2011-592), and Department of Veterans Affairs (1BX00090-01A1) to M.A.G.

所有程序被批准，并按照范德比尔特机构动物护理和使用委员会进行。 1.标签COI与荧光基团（可选） <ol><li>选择荧光染料，将与自由伯胺反应（ 例如 ，在一个蛋白质），如琥珀酰亚胺基酯或荧光素衍生物，可视化微球的货物。溶解8倍摩尔过量（相对于COI的摩尔数）的荧光团进入二甲基亚砜200微升（DMSO）中。 </li><li>悬浮50毫克COI高达800微升在车辆溶液的最终体积（62.5毫微克/微升的最终浓度）。车辆的解决方案将取决于COI的来源而变化。 注意:典型车辆溶液包括磷酸盐缓冲盐水（PBS）或DMSO中。 </li><li>从步骤1.1加入所有的荧光/ DMSO溶液，悬浮COI。涡旋过夜，在4℃。可替代地，涡旋在室温下将反应混合物（RT）下进行4小时。 </li><li>继标记反应，用脱盐柱去除多余的荧光。 <ol><li>从脱盐柱排水存储缓冲区，并以16毫升去离子水冲洗。通过丢弃所有流量。 </li><li>荧光标记COI添加到脱盐柱。 1.2毫升去离子水洗脱，并通过收集的流动。 </li><li>重复洗脱步骤的额外四次，每次加的1.2ml去离子水，并通过为单独的样品收集的流动。 </li><li>冷冻在-80℃下通过样品收集的所有流，并根据制造商的24小时指示冻干。 </li></ol></li></ol> 通过水包油包水乳液溶剂蒸发方法2. COI加载微球的制备<ol><li>加入1毫克的荧光标记的COI为100微升去离子水，形成第一水相（W1）。 </li><li>溶解65毫克聚（乳酸 - 共 - 乙醇酸ACID）（50:50丙交酯:乙交酯，分子量54000 - 在微量离心管中750μl的二氯甲烷69000）。 Ultrasonicate为10 - 30秒（在160瓦）以完全溶解的PLGA。这就形成了油相（O）。 </li><li>添加在步骤2.1向O相位在一个逐滴方式生成的W1相的所有。乳化使用手持式均化器在20,000rpm下30秒，以形成W1 / O相。 </li><li>所有在一个逐滴方式W1 / O相添加到15ml的1％（重量/体积）含水聚（乙烯醇）（PVA）溶液，并使用手持式均化器乳化以20,000rpm离心30秒。 </li><li>使用旋转蒸发1小时以除去溶剂和生成的水相的圆底烧瓶中，并受传送所有在步骤2.4中产生的乳液的一个200毫升至635毫米汞柱的真空。 </li><li>等分试样将1ml水相在步骤2.5到十四离心管生成。离心7500在微量离心管中的水溶液xg离心8分钟。 注:在此步骤中，微球存在于剩余的水溶液并浓缩至微量离心管的底部。 </li><li>小心地从离心管用微量取出900微升水溶液中，移液以便不打扰在微量离心管底部的微球。 <ol><li>通过加入1毫升去离子水于每个离心管中洗过量PVA的微球。离心机7500 xg离心8分钟，然后再次小心地从离心管用微量取出900微升水溶液中，移液以便不打扰在微量离心管底部的微球。 注:其余100μl的水溶液含有所生成的微球。 </li></ol></li><li>冻结在步骤2.7在-80℃，产生的水微球的解决方案。 </li><li>冷冻干燥的微球使用根据冻干机制造商的指令并贮存在-20℃。 注:通过完全溶解PLGA微球，并且相对于游离蛋白质7的标准曲线中的蛋白质浓度确定测量COI的装载效率。 </li><li>作为阴性对照，产生一批次的"空白"微球（以下简称为控制微球体）。 注意:控制微球的生成是相同的亲水COI-微球的产生，无COI除了800微升媒介物溶液中步骤1.2。相对于COI-PLGA微球对β细胞有丝分裂原测定PLGA质量浓度匹配控制颗粒。 </li></ol> 3.胰岛文化传媒和预试验培养基的制备<ol><li>制得200毫升介质完整小鼠胰岛的培养:RPMI补充有11毫米的葡萄糖，10％马血清，100U / ml青霉素G和100微克1640培养基/ ml链霉素（以下简称为让文化传媒）。 注1:与胎牛血清（FBS），马血清缺乏胎盘催乳素，β细胞增殖，混淆分析在增殖测定的诱导。 注2:由于PLGA微球不会在使用前消毒，在加入抗生素的培养基是至关重要的，以避免微生物污染。 </li><li>在50ml锥形管中的电子移液器和地点除去25毫升胰岛培养基。补充在50ml锥形管25毫胰岛培养基用0.2毫EGTA的终浓度，以产生预测定培养基。 注:加入EGTA轻度松动的胰岛细胞与细胞的接触，而不改变胰岛架构。这有助于确保COI可以到达胰岛细胞内，并有助于防止胰岛中央坏死。 </li></ol> 4.完整小鼠胰岛的培养<ol><li>从预先选定的应变，性别，AG隔离完好胰岛e和以下胰腺8,9的例行胶原酶消化小鼠的基因型。从游泳池样品一样一起小岛。 </li><li>等分试样40胰岛成在200μl胰岛培养基的96孔组织培养板的一个孔中。每口井目测尺寸匹配的胰岛（样本之间胰岛当量（IEQ）的精确评估是没有必要的）。填写紧邻井用200微升无菌水的小岛提供一个缓冲蒸发井空。在媒体过夜，在37℃培养胰岛95％空气和5％CO 2的气氛下。 </li></ol> COI装载和控制PLGA微球5.停牌重组<ol><li>过夜后胰岛的恢复，重新悬浮的微球通过将测定预媒体冻干COI-PLGA微球中的一个等分试样，使得COI中的离心管中最终浓度为10纳克/微升（COI存在于一个给定的量的量生成的万分之一pheres在步骤2.9）来测定。超声处理在冰水浴悬浮COI加载的微球在160 10分钟W和在4℃下10秒长的脉冲。 </li><li>悬浮控制PLGA微球通过将测定预介质的相同体积冻干控制PLGA微球的质量相等。超声处理在冰水浴再悬浮控制PLGA微球在160 10分钟W和在4℃下10秒长的脉冲。 </li><li>直观地确认微球通过吸取2微升两个玻璃盖玻片之间的微球悬浮分散。使用可视化上落射荧光或明显微镜40倍物镜球。 </li><li>如果微球显著聚合仍然可见，声处理，在冰水浴中在160额外10分钟W和在4℃下10秒长的脉冲和再悬浮的微球重复可视化。 </li></ol> 6.治疗COI加载或控制PLGA微球胰岛&lt;/ P&gt; <ol><li>对于每个孔与COI-微球被处理，稀释预先测定培养基再悬浮COI微球体的10毫微克/微升至100微升的最终体积和预定的最终浓度制备试验培养基。 注:该蛋白质的最优的最终浓度将对于用于该测定各COI变化。理想情况下，一个范围的最终浓度进行试验。 </li><li>每个孔用作对照，稀释在步骤5.2与用来稀释COI-微球在步骤6.1相同的稀释量产生的重悬的控制PLGA微球制备控制测定培养基。 注:与对照测定培养基处理的胰岛将作为阴性对照，以使空白微球体可具有对实验结果的任何影响的检测。 </li><li>小心地从每个孔中除去100μl的胰岛培养基用微量使得没有胰岛从孔脱落。轻轻地添加100μ升测定培养基或100微升控制试验培养基到每个孔中的。 </li><li>孵育在37℃的胰岛95％空气和5％CO 2的气氛下三天。 </li></ol> 7.完整分散到胰岛显微镜载玻片<ol><li>三天后，用微量小心从所有孔取出并丢弃试验介质以免撞出小岛。轻轻地加入200μl的PBS胰岛洗试验培养基他们。小心地取出，用微量丢弃PBS并轻轻地用一个额外的200微升PBS洗一遍小岛。 </li><li>捞出用微量弃PBS，加入100微升0.025％胰蛋白酶，2毫米EDTA溶液。孵育在室温3分钟。吸管胰岛中的胰蛋白酶-EDTA溶液上下每2分钟直到胰岛目视确认被分散成单个细胞（请注意，细胞的一些小块可能仍然存在）使用光学显微镜。转移每孔个体的整个体积LY到labeled-离心管。 </li><li>添加400μl的胰岛培养基到每个离心管中以停止胰蛋白酶介导的细胞解离。 </li><li>在4℃100 xg离心5分钟离心样品以沉淀胰岛细胞到离心管的底部。 </li><li>轻轻地取出用微量上清，重新悬浮颗粒在200微升新鲜胰岛培养基。 </li><li>使用CYTO离心机，离心机分离在140 XG 3分钟胰岛到充电显微镜载玻片。 </li><li>在RT风干载玻片10分钟，然后绘制围绕与疏水标记笔离解的胰岛一个框。 </li><li>修复用75微升4％低聚甲醛（PFA）在室温10分钟的细胞。除去4％PFA和在75微升轻轻洗涤细胞的PBS两次。洗涤后，将透用75微升0.2％的Triton X-100的PBS 10分钟的细胞。然后，立即用75微升PBS细胞。 注意:PFA是已知的过敏，致癌和有毒的。 </li></OL&gt; 8.游离的胰岛细胞免疫荧光标记胰岛素和细胞增殖标记，Ki67的<ol><li>通过将两个湿纸巾在100滑动容量显微镜滑动箱的底部制备潮湿室。 </li><li>广场幻灯片平倒在潮湿室（朝上细胞）。通过从使用微量先前洗涤步骤抽吸PBS并且加入阻断溶液的75微升准备用于标记样品（5％正常驴血清（NDS）的PBS），以每滑动阻止抗体与样品的非特异性结合。孵育载玻片在在RT 1小时封闭溶液。 </li><li>轻轻使用微量并添加75微升含豚鼠抗胰岛素和兔抗Ki67抗体初级抗体溶液抽吸封闭溶液，在PBS中稀释的1μg/ ml的5％NDS。孵育在RT 1小时。 注意:细胞增殖的替代标记包括增殖细胞核抗原（PCNA）与磷酸化ohistone H3（PH 3）， </li><li>使用微量轻轻吸一次抗体溶液和冲洗用75微升PBS中的细胞。吸出PBS中使用微量和冲洗细胞两次，用75微升的PBS各一次。孵育在在RT 1小时封闭液稀释至2微克/毫升75微升抗豚鼠Cy5的二级抗体和抗兔Cy3标记的二级抗体各样品。 注意:不要使用贴有一个已经用于标记微球相同或频谱重叠荧光第二抗体。 </li><li>使用微量并且在75微升为300nM的DAPI的PBS中在室温下孵育3分钟抽吸二级抗体溶液。抽吸DAPI使用微量并且在去离子水中漂洗5分钟。 </li><li>轻轻地从使用微量样品吸的水。现货滴（约100微升）载于玻璃盖玻片抗淬灭剂快干安装的解决方案。倒置显微镜载玻片样品的一面朝下，邻n要安装的解决方案。轻轻地用吸管尖除去气泡，擦干用柔软清洁纸巾多余的液体按盖玻片反对的幻灯片。允许盖玻片在RT干燥过夜。 </li></ol> 9.图像采集与分析<ol><li>使用落射荧光显微镜与连接的摄像头进行图像采集。对于每一个荧光团，确定最佳的曝光时间（通常为20毫秒DAPI，40 - 80毫秒胰岛素和250毫秒Ki67的）。确保图像采集参数相等对于所有样品。 </li><li>对于每个样本，算上人工胰岛素3000阳性细胞和那些算多少也Ki67的阳性。只计算有一个明确和清晰可见核胰岛素阳性细胞。计算通过分割的Ki67阳性/胰岛素阳性细胞的数目由胰岛素阳性细胞的总数再乘以100增殖β细胞对每个样品的百分比。 </li><li>天后etermining增殖β细胞对每个样品的百分比，确定是否控制和通过分析与使用市售统计软件方差单向结果实验样品之间存在的β细胞增殖的统计学显著差异。 </li></ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+-+a+novel+biomarker+for+DNA+double-strand+breaks.">Gamma-H2AX - a novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-309 (2008).</li><li>Daoud, J., Rosenberg, L., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+culture+and+preservation+strategies:+advances,+challenges,+and+future+outlook.">Pancreatic islet culture and preservation strategies: advances, challenges, and future outlook.</a> Cell Transplant. 19 (12), 1523-1535 (2010).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biol Proced Online. 11, 3-31 (2009).</li><li>Xu, Q., He, C., Xiao, C., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Oxygen+Species+(ROS)+Responsive+Polymers+for+Biomedical+Applications.">Reactive Oxygen Species (ROS) Responsive Polymers for Biomedical Applications.</a> Macromol Biosci. 16 (5), 635-646 (2016).</li><li>Makadia, H. K., Siegel, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+Lactic-co-Glycolic+Acid+(PLGA)+as+Biodegradable+Controlled+Drug+Delivery+Carrier.">Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier.</a> Polymers (Basel). 3 (3), 1377-1397 (2011).</li><li>Cui, F., Shi, K., Zhang, L., Tao, A., Kawashima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+loaded+with+insulin-phospholipid+complex+for+oral+delivery:+preparation,+in+vitro+characterization+and+in+vivo+evaluation.">Biodegradable nanoparticles loaded with insulin-phospholipid complex for oral delivery: preparation, in vitro characterization and in vivo evaluation.</a> J Control Release. 114 (2), 242-250 (2006).</li><li>Kavanaugh, T. E., Werfel, T. A., Cho, H., Hasty, K. A., Duvall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-based+technologies+for+osteoarthritis+detection+and+therapy.">Particle-based technologies for osteoarthritis detection and therapy.</a> Drug Deliv Transl Res. , (2015).</li><li>Joshi, R. V., Nelson, C. E., Poole, K. M., Skala, M. C., Duvall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+pH-+and+temperature-responsive+microparticles+for+protein+delivery+to+ischemic+tissues.">Dual pH- and temperature-responsive microparticles for protein delivery to ischemic tissues.</a> Acta Biomater. 9 (5), 6526-6534 (2013).</li><li>Poole, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS-responsive+microspheres+for+on+demand+antioxidant+therapy+in+a+model+of+diabetic+peripheral+arterial+disease.">ROS-responsive microspheres for on demand antioxidant therapy in a model of diabetic peripheral arterial disease.</a> Biomaterials. 41, 166-175 (2015).</li></ol>

The authors have nothing to disclose.

β细胞增殖的培养物研究通常是由几个困难阻碍。首先，永生化β细胞系由较高程度的增生比什么是内源性β细胞活胰岛发现的特点。此外，这些永生化细胞系缺乏正常结构正常β细胞功能的关键。这两个事实使得难以确定是否使用永生化的β细胞系在体内或整个胰岛进行测试时将保持真获得的结果。我们描述的方案，它使用新鲜分离完整小鼠胰岛，如胰岛架构被保持和β细胞增殖相当于?...

胰岛β细胞是在体内仅产生胰岛素的细胞，并维持血糖稳态的关键。而健康个体具有足够的β细胞量和功能适当地调节血糖，糖尿病的个体被不足β细胞量和/或功能1,2-特征。已经提出了诱导β细胞增殖可以最终增加β细胞量和个体的糖尿病3恢复葡萄糖动态平衡。然而，评价和完整的胰岛β潜能细胞增殖的化合物的验证是必要的，可以制定有效的治疗之前。尸体人胰岛与糖尿病患者的移植恢复血液葡萄糖稳态了一段时间，但是可用性和此实验过程的成功是由可供移植人胰岛的缺乏和由β细胞死亡中的胰岛船尾受阻呃移植4。即使使用的诱导胰岛素产生细胞的倍增因子的发现，一个重大的挑战仍然存在于提供这些因素的体内相关网站。对于持续局部递送β细胞增殖的化合物的一个策略是聚（乳酸 - 共 - 乙醇酸）（PLGA）。 PLGA在FDA的使用的历史认可药物递送制品，由于其高的安全性，生物降解性，并且延长释放动力学5。具体地，PLGA是通过水解与水在体内或在培养成乳酸和乙醇酸，其天然存在于体内存在的代谢产物降解的丙交酯和乙交酯的共聚物。包封的药物化合物可以通过扩散和/或降解受控释放机构周围环境释放。 COI的封装提供了保护，防止酶降解，提高试剂的生物利用度相比unencapsula特德COI 5。我们建议，PLGA微球能够最终在体内用于施用在培养候选化合物对完整的胰岛和。测试PLGA的功效来管理β细胞有丝分裂原到胰岛离体进行了探索移植协议之前是至关重要的。 目前，没有任何技术来测量在活的动物的β细胞增殖。实验评估体内潜在的增殖的化合物的有效性，因此需要这些化合物的行政生活的动物，为胰腺免疫标记随后的解剖和处理。这些协议是昂贵和费力的，并且所需要的化合物要全身施用，不作任何保证，他们将到达小岛。相反地​​，几种永生β细胞系可用于在培养产生胰岛素的细胞的研究，但这些细胞系缺乏胰岛架构和环境下吨生物6中。永生化β细胞系也被表征为具有高得多的程度比内源性β细胞在体内的复制，从而变得复杂诱导增殖的化合物的分析。在这项研究中，我们描述了使用从成年小鼠中分离的完整的胰岛的协议。与β细胞系，完整保留胰岛胰岛的正常结构。同样，在对比体内进行的试验中，给予增殖性化合物直接向培养的完整胰岛显著减少了必须精确地测量β细胞增殖的试剂的量。 目前的研究利用PLGA来管理COI，在这个例子中，重组人结缔组织生长因子（rhCTGF）。此处所描述的方法赋予通过原始化合物培养的胰岛给药的显著优势，因为它允许对一个持续释放化合物的成第e媒体。值得注意的是，该测定可以被修饰以管理各种蛋白质和感兴趣的完整胰岛抗体。在其他内分泌细胞类型，包括α-细胞的作用，也可以被分析。

<table><tbody><tr><td>Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td><td>ThermoFisher Scientific</td><td>O6149</td><td>For labeling COI with fluorophore</td></tr><tr><td>DMSO Dimethyl Sulfoxide</td><td>Fisher BioReagents</td><td>BP231-1</td><td>For dissolving fluorophore in step 1</td></tr><tr><td>Disposable PD-10 Desalting Columns</td><td>GE Healthcare</td><td>17-0851-01</td><td>Desalting column used in step 1</td></tr><tr><td>Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000 - 69,000</td><td>Sigma-Aldrich</td><td>739960</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>Poly(vinyl alcohol) molecular weight 89,000 - 98,000</td><td>Sigma-Aldrich</td><td>341584</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>RPMI 1640</td><td>Thermo Scientific</td><td>11879-020</td><td>For culturing islets</td></tr><tr><td>Dextrose Anhydrous</td><td>Fisher BioReagents</td><td>200-075-1</td><td>Supplement for islet media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>Antibiotics for islet media</td></tr><tr><td>Normal horse serum</td><td>Jackson ImmunoResearch</td><td>008-000-121</td><td>Supplement for islet media</td></tr><tr><td>96-well tissue culture plate</td><td>Corning</td><td>3603</td><td>For culturing islets</td></tr><tr><td>Ethylene glyco-bis(2-aminoethylether)-&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E4378</td><td>Supplement for pre-assay islet media</td></tr><tr><td>Cytospin 4 Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>For spinning cells onto microscope slides</td></tr><tr><td>EZ Single Cytofunnel</td><td>Thermo Scientific</td><td>A78710020</td><td>For spinning cells onto microscope slides</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher BioReagents</td><td>BP118-500</td><td>Used in dissociating islets</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>For fixing cells</td></tr><tr><td>Triton&amp;nbsp; X-100</td><td>Fisher BioReagents</td><td>BP151</td><td>For permeabilizing cells</td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td>Blocking reagents for immunofluorescence</td></tr><tr><td>Anti-Ki67 antibody</td><td>abcam</td><td>ab15580</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Polyclonal Guinea Pig Anti-Insulin</td><td>Dako</td><td>A0564</td><td>For insulin immunofluorescence</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Guinea Pig</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td>For insulin immunofluorescence</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>ThermoFisher Scientific</td><td>D1306</td><td>For nuclei visualization in immunofluorescence</td></tr><tr><td>Aqua-Mount</td><td>Lerner Laboratories</td><td>13800</td><td>Fast drying mounting media</td></tr><tr><td>FreeZone -105 &amp;deg;C 4.5 Liter Cascade Benchtop Freeze Dry System</td><td>Labconco</td><td>7382020</td><td>For lyophilization of microspheres</td></tr></tbody></table>

sustained administration of &#946;-cell mitogens to intact mouse islets ex vivo using biodegradable poly(lactic-co-glycolic acid) microspheres

生物材料的发展已显著增加用于靶向药物递送的潜在的各种细胞和组织类型，包括在胰岛β细胞。此外，生物材料颗粒，水凝胶，和支架还提供了一个独特的机会来管理持续的，可控的药物输送到β细胞的培养和移植的组织模型。这些技术允许使用完整的小岛和一个翻译相关制度的候选β细胞增殖因子的研究。此外，确定用于在培养系统刺激β细胞增殖的候选因素的有效性和可行性是向前移动到体内模型之前的关键。这里，我们描述的方法共培养完整小鼠胰岛与感兴趣的可生物降解化合物（COI）-loaded聚（乳酸-共-乙醇酸）（PLGA）微球，以评估原位释放ö持续的影响的目的˚F促有丝分裂因子对β细胞的增殖。这种技术详细描述了如何生成包含使用市售的试剂所需的货物PLGA微球。而所描述的技术使用的重组人结缔组织生长因子（rhCTGF）作为一个例子，可以很容易地用于各种COI的。此外，此方法利用的96孔板，以尽量减少必要评估β细胞增殖的试剂的量。该协议可以很容易地适合于使用替代生物材料和其它内分泌细胞特征，例如细胞存活和分化的状态。

图1是利用上述协议所产生的微球的视觉表示。这里所描述的协议产生各种大小的rhCTGF加载的微球。微球体的最大分数为1和10微米之间的直径，尽管一些微球可以更大（ 图 2）。如果需要的话，微球尺寸可以被调整，并根据工艺参数，如均化速度和时间，所用表面活性剂浓度，并且每个水/油/水相10的相对体积进行优化。 <p ...

Watch this Scientific Journal Video about Sustained Administration of Î²-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Polylactic-co-glycolic acid Microspheres at JoVE.com

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

sustained-administration-cell-mitogens-to-intact-mouse-islets-ex-vivo

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Vanderbilt University Medical Center. Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.

视频: Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

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

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

胰岛血气 StarPEG Nanocoating 的表面工程

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

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 ...

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 ...

生物学

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

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

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

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

体外胰腺器官从分散的小鼠胚胎祖细胞

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

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 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.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

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 ...

β细胞的有丝分裂原持续管理局完整小鼠胰岛

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