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细胞增殖相当于在体内发现的规避了这些问题。 培养完整胰岛当一个显著关注的是，胰岛芯内的细胞将经历缺氧诱导坏死或不会暴露到rhCTGF的可能性。因为这个原因，最终为0.1mM的EGTA浓度被添加到媒体松开细胞 - 细胞接触，而不破坏胰岛架构。我们先前已经公布了单独加入EGTA可以增加β细胞的增殖，这可能是由于在媒体上的胰岛芯12的营养物质和有丝分裂因子的增加的访问。响应于EGTAβ细胞增殖的增加也可能是由于在细胞-细胞接触本身13,14下降。在这个协议中所描述的媒体也被补充有马血清代替更传统的胎牛血清。这个取代是由于胎盘催乳素在胎牛血清存在下，其可以刺激自身β细胞的增殖，在增殖测定潜在复杂分析的。 的PLGA微球（有或没有COI）至胰岛细胞的相对毒性的测定可以通过马的免疫荧光分析来评估细胞死亡或DNA损伤的rkers包括TUNEL或γ-H2AX 15,16。虽然除了PLGA微球的未示出任何明显的不利影响，以胰岛离体 ，用户应该知道的微球可具有图像分析的影响。如在图3显示出，并在结果部分所提到的，未降解的微球可以是如免疫荧光图像中的黑斑，同被用于增加浓度时出现更多的微球明显。 应当指出的是，描述的协议已针对具有隔离小鼠胰岛工作。因此，目前还不清楚，如果在相同的条件将与来自替代的生物，如鼠和人类收获胰岛工作。值得注意的是，胰岛收获来自不同生物的通常具有不同的最佳培养条件和不同程度的β细胞增殖17,18。 许多BIOM材料板是潜在可用的施用rhCTGF到分离的胰岛，包括聚（酮缩硫醇-氨基甲酸酯）和聚（硫化丙烯）19。我们选择把重点放在PLGA由于它具有几个显着的特质。首先，PLGA是一个比较实惠的试剂和微球可以用可在大多数研究机构的标准设备（离心机，均质机，冻干机）产生。 PLGA是有由于其生物相容性，生物可降解性，其调节化合物的释放率20能力，FDA批准的设备成功使用先例的人造聚合物。 PLGA降解通过水解在水的存在下，在此过程中释放出的货物。 PLGA粒子的化学组成可以在一化合物在体内在几个星期的期间释放量身定做这样。 PLGA也已在临床前动物试验和人临床疗法用于局部给药的化合物对各种器官和Tissu酒店ES 21,22。其它疏水性聚合物也可以在可生物降解的微球的生成中使用。这些聚合物可以以特定环境刺激，例如pH，温度，和活性氧23,24的存在作出响应。因此，研究人员应仔细考虑其生物材料是最适合他们的学业。 任何利用PLGA，或其他生物材料研究，以研究β细胞增殖的体外应该意识到包封COI的相比未包封COI效果之间的电位差。例如，通过PLGA rhCTGF的递送可以相比于用未包囊蛋白治疗改变的β细胞增殖的诱导的程度和时间。目前在我们的实验室正在进行的研究正在研究这些潜在的分歧。 β细胞增殖的培养胰岛的分析是识别和我一个强大的模型β细胞的有丝分裂原的chanistic分析。使用此处描述我们显示用于与延长释放递送载体给药COI分离的培养的胰岛相对快速且经济有效的方法测定。该测定应该纯粹是根据它的先前公布的刺激β细胞增殖的体内能力和离体 11是适用于感兴趣几乎任何COI，和我们的选择集中于CTGF。此外，该法可推进到胰岛在活的生物体移植模型之前验证的有效性和使用PLGA作为传递方法的安全性。总体而言，所描述的协议提供了施用化合物培养的胰岛与测量PLGA的安全可移植组织一个更广泛的影响的新方法。

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 border="0" cellpadding="0" cellspacing="0" width="819">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">O6149</td>
			<td style="width:307px;">For labeling COI with fluorophore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO Dimethyl Sulfoxide</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP231-1</td>
			<td>For dissolving fluorophore in step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;">17-0851-01</td>
			<td>Desalting column used in step 1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000-69,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">739960</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(vinyl alcohol) molecular weight 89,000-98,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">341584</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">11879-020</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose Anhydrous</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">200-075-1</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td>Antibiotics for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal horse serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">008-000-121</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well tissue culture plate</td>
			<td style="width:179px;">Corning</td>
			<td align="right" style="width:119px;">3603</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethylene glyco-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td>Supplement for pre-assay islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytospin 4 Cytocentrifuge</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78300003</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EZ Single Cytofunnel</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78710020</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td>BP118-500</td>
			<td>Used in dissociating islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>For fixing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton&#160; X-100</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP151</td>
			<td>For permeabilizing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal donkey serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">017-000-121</td>
			<td>Blocking reagents for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Ki67 antibody</td>
			<td style="width:179px;">abcam</td>
			<td style="width:119px;">ab15580</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyclonal Guinea Pig Anti-Insulin</td>
			<td style="width:179px;">Dako</td>
			<td style="width:119px;">A0564</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy3 AffiniPure Donkey Anti-Rabbit</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">711-165-152</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy5 AffiniPure Donkey Anti-Guinea Pig</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">706-175-148</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>For nuclei visualization in immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aqua-Mount</td>
			<td style="width:179px;">Lerner Laboratories</td>
			<td align="right" style="width:119px;">13800</td>
			<td>Fast drying mounting media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">FreeZone -105&#176;C 4.5 Liter Cascade Benchtop Freeze Dry System</td>
			<td style="width:179px;">Labconco</td>
			<td align="right" style="width:119px;">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。 以下与PLGA微球和随后的免疫标记处理完好胰岛的扩散，这是经常可以看到样品不含细胞（ 图3）之间的任何标记的区域。虽然大多数是仍然完好无损培养治疗期后的微球是在洗涤步骤去除，一些残留的胰岛旋涂在显微镜载玻片后。这些微球导致成像期间可视孔状结构。这些残留的微球通常不与随后的量化干扰。 成像后，Ki67的阳性/胰岛素阳性细胞的百分比可以通过手动计数标记的细胞的总数，或使用软件的图像分析来定量。此前，我们已经证明，重组人结缔组织生长因子（rhCTGF）能刺激小鼠胰岛β细胞增殖，胰岛完整体外 11。使用上述协议中，我们生成了包含rhCTGF（rhCTGF-PLGA）微球。治疗完好胰岛与rhCTGF-PLGA微球3天导致β细胞增殖有类似的增加与前面的原始蛋白的报道，这表明该蛋白没有罗<html>瑟微球生成（ 图4）中的任何功能。 <img alt="图1" src="/files/ftp_upload/54664/54664fig1.jpg" /> 图1:PLGA微球的可视化表示通过将荧光染料到制造协议，PLGA微球可使用表面荧光显微镜来观察。比例尺代表200微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54664/54664fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54664/54664fig2.jpg" /> 图2:PLGA微球的粒度分布虽然尺寸分布可以变化，大多数的微球将小于10微米的直径。ARGET ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54664/54664fig3.jpg" /> 图 3: 分散相增殖β细胞免疫荧光可视化分散胰岛免疫标记的增殖标记Ki67的（红色）和胰岛素（绿色）来标记增殖β细胞。细胞核被标以DAPI（蓝色）。箭头指示的Ki67阳性/胰岛素阳性细胞。星号（*）表示未降解微球。比例尺为100μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54664/54664fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54664/54664fig4.jpg" /> 图4: 量化与分析β细胞临liferation。胰岛具有不同量rhCTGF-PLGA微球处理。的Ki67阳性/胰岛素阳性细胞的数目是手动从所有样品计算并表示为胰岛素阳性β细胞总数的百分比。 X轴对应于rhCTGF存在于各处理的最终浓度。使用单向ANOVA，然后通过Tukey多重比较检验，确定统计学意义。统计显着性设定为p≤0.05。误差棒代表平均值的标准误差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54664/54664fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sustained Administration of ÃŽÂ²-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

Nanyang Technological University

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

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

体外拟态正常与异常的人造血

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

光频域成像<em&gt;体外</em肺切除标本:获得一对一的图像病理组织学的相关性

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

可生物降解聚合物颗粒的淋巴内结注射

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...