我们要感谢我们的很多同事都在项目的各个阶段提供帮助，建议和重要的投入。制作这个视频文章支持资金从德国教育和研究部（BMBF）德国糖尿病研究（DZD，中心部<a href="http://www.dzd-ev.de">http://www.dzd-ev.de</a> ），IMIDIA（ <a href="http://www.imidia.org">http://www imidia.org</a> ）和大学医院的卡尔古斯塔夫Carus在德累斯顿工业大学。导致这些结果的研究，已收到来自欧洲共同体的第七框架计划（FP7/2007-2013）的资助创新药物倡议下赠款协议N ° 115005。

1。胰腺组织收集在手术室<ol><li>外科医生执行的胰腺部分切除。 </li><li>胰腺标本放置在一个盒子上冰后，立即送到病理学家。 </li></ol> 2。胰岛隔离的组织选择<ol><li>病理学家选择柔软，健康的组织出现，保留用于诊断目的的受损组织。提供不少于2克可用组织的纤维组织和标本排除胰岛隔离。 </li><li>沉浸在欧元Collins液的胰腺组织和实验室提供的冰。 </li></ol> 3。人类胰岛的分离<ol><li>称取胰腺组织，然后放入一个10厘米的菜。 </li><li>在500毫升容量瓶中放入150毫升的RPMI媒体。 </li><li>在250毫升容量瓶中，准备130毫升的RPMI媒体相结合，与100毫克/毫升DNase和20毫升5mg/ml Liberase RI的消化酶活性的解决方案。绘制成一个注射器注入胰腺组织10毫升这种解决方案。 </li><li>注入胰腺组织消化酶的解决方案，旨在它均匀扩张。如果这是防止纤维化，消化，很可能会失败。下一步，组织3〜4毫米到百果冰块。 </li></ol> 4。人的胰腺消化电路<ol><li>组装消化电路如图1所示。 Ricordi腔中的流动方向是在出室顶部和底部。 </li><li>其余消化酶液500毫升容量瓶中，加入到300毫升的总量，并插入温度计。 </li><li>室转移到胰腺组织，插入目（孔径为600微米）和氮化硅大理石（直径：15毫米），接近香港总商会和启动泵与定为140毫升/分钟的流量。 </li><li>当电路的温度达到37℃，开始计时，并定期抽取样本，以确定何时停止消化。 （2毫克/毫升）双硫腙染色允许微观可视化的胰岛细胞内消化的组织 4 。 </li><li>一旦胰岛周围的腺泡组织分开，迅速停止加热线圈放置在冰上，加入200毫升（900毫升的RPMI 1640与5.5毫米的葡萄糖和10％FBS）冷洗媒体电路消化。 </li><li>收集在250毫升的锥形管胰岛解决方案，因为它散发出来的样品管。继续，直到电路是空的。 </li></ol><img alt="图1" src="/files/ftp_upload/2962/2962fig1.jpg" /> 图1。人类胰腺消化电路人类胰腺消化电路包括一个Ricordi腔目（孔径为600微米）和三个氮化硅大理石（直径：15毫米） 。从室流出驱动组织收集瓶中，由蠕动泵（140毫升/分）。箭头表示水流方向。酶消化的最佳温度是保持浸泡在37℃水浴加热线圈。 5。洗涤后的消化<ol><li>离心机含有胰岛解决方案，在1000 rpm，4 ° C 5分钟250毫升锥形管。 </li><li>弃上清，重悬（900毫升与5.5毫米的葡萄糖和10％胎牛血清的RPMI 1640）洗媒体胰岛颗粒，分布在50 ml锥形管同等分数。 （可选：分发到额外的锥形管，以提高洗涤）离心机在1000 rpm，4℃5分钟。 </li><li>弃上清，轻轻地松开手册晃动的颗粒。 </li></ol> 6。在聚蔗糖梯度的分离纯化<ol><li>重悬浮颗粒与聚蔗糖媒体密度1.125克/毫升，pH值7.4，慢慢的覆盖密度为1.080，1.060和1.037克/毫升的聚蔗糖媒体10毫升分装。 </li><li>在2400转离心聚蔗糖梯度，4℃20分钟。 </li></ol> 7。胰岛收集和文化<ol><li>离心后，可以区分不同的组织成分，其分区，在渐变。 </li><li>弃上层脂肪和结缔组织组成。 </li><li>仔细收获胰岛总量在1.037-1.060克/毫升相间的第一层。这一层包含了最纯粹的孤立的小岛。收集分数分别为有效地隔离。 </li><li>收集第二，胰岛总量少纯在1.060-1.080克/毫升相间的一小部分。 </li><li>加入洗涤介质（900毫升与5.5毫米的葡萄糖和10％胎牛血清的RPMI 1640）每个锥形管总量为50毫升稀释的聚蔗糖梯度。离心机在1000 rpm，4℃5分钟。 </li><li>丢弃吸上清。使用的颗粒可能会有所松动，由于聚蔗糖梯度的照顾。 </li><li>洗颗粒无线媒体日洗（900毫升5.5毫米的葡萄糖和10％FBS）的RPMI 1640和1000转的离心机，4 ° C加热5分钟。重复使用胰岛文化传媒</li><li>在培养基重悬的小岛，并放置在孵化器，用5％的CO 2在37℃为24-48小时，然后再进一步加工彗星。 </li></ol>

<ol>
	<li>Kahn, S. E., Zraika, S., Utzschneider, K. M., Hull, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+cell+lesion+in+type+2+diabetes:+there+has+to+be+a+primary+functional+abnormality.">The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.</a> Diabetologia. 52, 1003-1012 (2009).</li><li>Deng, S., Vatamaniuk, M., Huang, X., Doliba, N., Lian, M. M., Frank, A., Velidedeoglu, E., Desai, N. M., Koeberlein, B., Wolf, B., Barker, C. F., Naji, A., Matschinsky, F. M., Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+islets+isolated+from+type+2+diabetic+subjects.">Structural and functional abnormalities in the islets isolated from type 2 diabetic subjects.</a> Diabetes. 53, 624-632 (2004).</li><li>Ricordi, C., Lacy, P. E., Finke, E. H., Olack, B. J., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+method+for+isolation+of+human+pancreatic+islets.">Automated method for isolation of human pancreatic islets.</a> Diabetes. 37, 413-420 (1988).</li><li>Latif, Z. A., Noel, J., Alejandro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+staining+fresh+and+cultured+islets.">A simple method of staining fresh and cultured islets.</a> Transplantation. 45, 827-830 (1988).</li><li>Ehehalt, F., Knoch, K., Erdmann, K., Krautz, C., J&#228;ger, M., Steffen, A., Wegbrod, C., Meisterfeld, R., Kersting, S., Bergert, H., Kuhlisch, E., Bornstein, S., Bonifacio, E., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+turnover+in+islets+from+type+2+diabetic+patients.">Impaired insulin turnover in islets from type 2 diabetic patients.</a> Islets. 2, 30-36 (2010).</li><li>Hardt, P. D., Brendel, M. D., Kloer, H. U., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pancreatic+diabetes+(type+3c+diabetes)+underdiagnosed+and+misdiagnosed?.">Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed?.</a> Diabetes Care. 31, S165-S169 (2008).</li><li>Meisterfeld, R., Ehehalt, F., Saeger, H. D., Solimena, 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+disorders+and+diabetes+mellitus.">Pancreatic disorders and diabetes mellitus.</a> Exp Clin Endocrinol Diabetes. 116, S7-S12 (2008).</li><li>Verge, C. F., Gianani, R., Kawasaki, E., Yu, L., Pietropaolo, M., Jackson, R. A., Chase, H. P., Eisenbarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+type+I+diabetes+in+first-degree+relatives+using+a+combination+of+insulin,+GAD,+and+ICA512bdc/IA-2+autoantibodies.">Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies.</a> Diabetes. 45, 926-933 (1996).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Marselli, L., Thorne, J., Ahn, Y. B., Omer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clin. Endocrinol. Metab. 93, 1046-1053 (2008).</li></ol>

没有利益冲突的声明。

使用我们的协议，可以分离出人类胰岛收集从部分胰腺切除胰腺组织。此协议的成功依赖于在几个关键点，采取的护理。为了保护β-细胞的活力，它是必不可少的，迅速运冰实验室标本。此外，必须优化组织消化的时间经验，根据组织纤维化和媒体的酶的活性等级。这也是真正的手动应用Ricordi腔的机械力的程度。因此，取得了良好的胰岛产量，该协议是最好由一个专门的科学家或技术助理。最初的失败是常态，除非球队已经经历胰岛隔离。 有我们的胰岛隔离协议和标准的人类胰岛隔离方法之间的主要区别：1）虽然我们使用在手术过程中受到几个小时的缺血胰腺组织，它是在现场立即处理。这是相反胰腺从胰腺/胰岛移植，这仍然分配和交付胰岛隔离设施过程中的几个小时的缺血，脑死亡捐赠者explanted的。 2）我们直接注入胶原酶的胰腺组织，而标准的协议注入胰管。 3）我们单独使用间断聚蔗糖梯度，而不是使用COBE Cell处理器从一个连续的聚蔗糖梯度收集胰岛的胰岛细胞。 手术标本是太纤维化或隔离胰岛足够数量的稀缺的情况下，组织仍可以检索通过激光捕获显微切割（LCM）的10。这使得基因表达数据恢复从几乎每一个标本，即使胰岛隔离失败或产量很低。不幸的是，液晶显示模块不生产活细胞功能研究，其金额通常是不足的蛋白质组学分析。因此，在使用LCM与我们的胶原酶消化协议平行检索胰岛可能是最有效的方法来处理手术切除标本。 收集部分pancreatectomies胰岛分离组织时，重要的是要仔细检查病人的临床病史和代谢状态。 3C型糖尿病，即糖尿病继发胰腺疾病导致手术6，部分pancreatectomized患者可能会受到影响。 43参与我们的部门在2010年接受这种手术的患者中，32名非糖尿病患者，5例2型糖尿病患者和6影响类型3C糖尿病。同意这些数据与以往的研究表明糖代谢受损和糖尿病的一个相当大的患者患有胰头癌或慢性胰腺炎6部分。我们认为，糖尿病的主要原产地，如果被诊断至少一年之前的症状，导致胰腺手术7发病。胰岛自身抗原抗体水平也应测定，以评估潜在的自身免疫性糖尿病8来源。由于病人接受胰腺切除可能遭受从确诊糖尿病或葡萄糖不耐，应给予所有非糖尿病患者口服葡萄糖耐量试验之前，胰腺切除术9。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th></tr><tr><th>设备</th><th></th><th></th></tr><tr><td> 1毫升血清移液器（蜂星） </td><td>格雷纳生物一</td><td> 604181 </td></tr><tr><td> 10毫升血清移液器（蜂星） </td><td>格雷纳生物一</td><td> 607180 </td></tr><tr><td> 25毫升血清移液器（蜂星） </td><td>格雷纳生物一</td><td> 760180 </td></tr><tr><td> 10 ml注射器</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 300912 </td></tr><tr><td> 50 ml注射器</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 300865 </td></tr><tr><td> 5 ml注射器</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 309050 </td></tr><tr><td> 18 G 1,1 / 2针Microlance 3 </td><td>屋宇署（流式细胞Dickinson公司） </td><td> 304622 </td></tr><tr><td> 27 GX1 / 2针</td><td>布劳恩</td><td> 4658300 </td></tr><tr><td> 27 GX3 / 4针Microlance 3 </td><td>屋宇署（流式细胞Dickinson公司） </td><td> 302200 </td></tr><tr><td> 3厘米细胞培养皿中的2x2毫米电网</td><td> NUNC </td><td> 174926 </td></tr><tr><td> 20 cm组织培养皿</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 353003 </td></tr><tr><td> 6厘米轻松好握的培养皿</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 351016 </td></tr><tr><td> 150毫升储存瓶</td><td>康宁公司</td><td> 431175 </td></tr><tr><td> 250毫升的锥形瓶</td><td>肖特杜兰</td><td> 21 217 36 </td></tr><tr><td> 500毫升锥形瓶</td><td>肖特杜兰</td><td> 21 217 44 </td></tr><tr><td> 250毫升螺帽锥底离心管</td><td>康宁公司</td><td> 430776 </td></tr><tr><td> 50毫升猎鹰锥形管</td><td>屋宇署（流式细胞Dickinson公司） </td><td> 352070 </td></tr><tr><td> 260毫升Easyflask非治疗</td><td> NUNC </td><td> 156800 </td></tr><tr><td> Millex GV过滤单元Duropore（0.22微米） </td><td> Millipore公司</td><td> SLGV033RB </td></tr><tr><td>酒瓶上方的真空过滤机（PES的70毫米直径卷材，0.22微米孔径，45毫米脖子大小，500 mL体积） </td><td>康宁公司</td><td> 431118 </td></tr><tr><td>密度仪（Densito 30PX）LWE37463 </td><td>梅特勒 - 托利多</td><td> 51324450 </td></tr><tr><td>快速PES过滤单元0.2微米</td><td> NALGENE </td><td> 569-0020 </td></tr><tr><td>加热线圈</td><td> BioRep技术公司</td><td> HC - 02 </td></tr><tr><td> Multifuge 4 KS - R </td><td>贺利氏</td><td> 75015680 </td></tr><tr><td>泵典型PD5206 </td><td> Heidolph </td><td> 523-52060-00-2 </td></tr><tr><td> Ricordi室（600微米孔径的金属网状） </td><td> BioRep技术公司</td><td>型号的U - 50 - 01，串行0503-001 </td></tr><tr><td>制备架</td><td> BioRep技术公司</td><td> 50 - PS - 01 </td></tr><tr><td> O形圈</td><td> BioRep技术公司</td><td> OR - 50，U - 01 </td></tr><tr><td>氮化硅大理石9毫米（15集） </td><td> BioRep技术公司</td><td> SN - 01 </td></tr><tr><td>硅套管</td><td>聚乙烯</td><td> ř 3603 8,0 X4，0毫米</td></tr><tr><td>手术钳</td><td>布劳恩</td><td> BD168R </td></tr><tr><td>手术剪（蛇牌） </td><td>布劳恩</td><td> BC273R </td></tr><tr><td>水浴OLS是200 </td><td>津贴</td><td> OLS的200 </td></tr><tr><th>试剂</th><th></th><th></th></tr><tr><td>胶原酶 - &gt; Liberase RI（100毫克） </td><td>罗氏公司</td><td> 11815032001 </td></tr><tr><td> D（+）-葡萄糖</td><td>默克公司</td><td> 1.08337.1000 </td></tr><tr><td>二甲基亚砜（DMSO） </td><td>默克公司</td><td> 1.16743.1000 </td></tr><tr><td>二苯基卡巴腙（DTZ） </td><td>西格玛爱秩序</td><td> D5130 </td></tr><tr><td>我DNA酶（100毫克） </td><td>罗氏公司</td><td> 10104159001 </td></tr><tr><td> Dulbeccos 1xPBS </td><td>临时机场管理局实验室</td><td> H15 - 002 </td></tr><tr><td> Electrolytsolution E154 </td><td> Serumwerk </td><td> 00509 </td></tr><tr><td>胎牛血清（FBS） </td><td>临时机场管理局实验室</td><td> A15 - 101 </td></tr><tr><td>聚蔗糖400 </td><td>西格玛爱秩序</td><td> F9378 </td></tr><tr><td>胎儿牛血清（FBS） </td><td> Gibco公司</td><td> 10270-106 </td></tr><tr><td> 2 - （4 - （2 - 羟乙基） - 1 - 哌嗪基）ethansulfons‰URE（HEPES） </td><td>罗斯</td><td> 9105.3 </td></tr><tr><td>氢氧化钠，5米</td><td>临床药学，医院</td><td> - </td></tr><tr><td>青霉素/链霉素（100倍） </td><td>临时机场管理局实验室</td><td> P11 - 010 </td></tr><tr><td> Pipettaid（快递） </td><td>屋宇署（流式细胞Dickinson公司） </td><td> 357591 </td></tr><tr><td> Potassiumchlo​​rid </td><td> Fluka公司</td><td> 60132 </td></tr><tr><td> Potassiumdihydrogenphospate无水</td><td> JT贝克</td><td> 0241 </td></tr><tr><td> Potassiummonohydrogenphosphate </td><td> JT贝克</td><td> 3246-01 </td></tr><tr><td>中等的RPMI 1640 [ - ] D -葡萄糖[+] L -谷氨酰胺</td><td> Gibco公司</td><td> 11879-020 </td></tr><tr><td>中等的RPMI 1640 [+] L -谷氨酰胺</td><td>临时机场管理局实验室</td><td> E15 - 840 </td></tr><tr><td> Sodiumhydrogencarbonat </td><td>默克公司</td><td> 1.06329.0500 </td></tr></tbody></table> 媒体和解决方案双硫腙解决方案<ul><li>溶解在10毫升DMSO戴德梁行100毫克和40毫升PBS稀释</li><li>无菌过滤解决方案（0.22微米） </li></ul>酶液<ul><li>溶解100毫克，20毫升的RPMI 1640媒体Liberase RI（无补充） </li><li> 100毫克DNA酶溶解于1毫升的RPMI 1640介质（没有补充） </li><li>无菌过滤解决方案（0.22微米），并保持在冰上酶</li></ul>洗净媒体（1000ml/5.5mM葡萄糖） <ul><li>溶于100毫升FBS（热灭活） </li><li>添加450毫升RPMI 1640培养液（无葡萄糖） </li><li>添加450毫升的RPMI 1640（11毫米葡萄糖） </li><li>无菌过滤解决方案（0.22微米） </li></ul>胰岛文化传媒（500毫升/ 5,5毫米葡萄糖） <ul><li>溶于10毫升1M HEPES（pH值7.4） </li><li>添加50毫升胎牛血清</li><li>添加5 ml青霉素/链霉素（100倍） </li><li>添加2.75毫升1个M葡萄糖溶液</li><li>添加432.25毫升的RPMI 1640（无葡萄糖） </li><li>无菌过滤解决方案（0.22微米） </li></ul>欧元柯林斯解决方案<ul><li> 4,083克钾磷酸氢</li><li> 70,0 g葡萄糖</li><li> 2,237克氯化钾</li><li> 14,80克钾monohydrogenphosphate </li><li> 1,680克钠碳酸氢盐</li><li> 40毫升的电解质溶液（E 154） </li><li> pH值7.4 </li><li>加蒸馏水至2000毫升</li><li>无菌过滤解决方案（0.22微米） </li></ul>聚蔗糖库存解决方案<ul><li>加入欧元Collins液1000毫升至500克聚蔗糖</li><li>添加8.94克HEPES </li><li>搅拌过夜，直到聚蔗糖是解决</li><li> pH值7.4 </li><li>测量的密度</li></ul>聚蔗糖梯度<ul><li>根据密度计算的FBS，欧元Collins液和聚蔗糖库存解决方案</li><li>结合3解决方案，并用密度计测量的密度</li><li>欧元柯林斯解决方案或聚蔗糖库存解决方案，直到密度调整</li><li>无菌过滤器的解决方案（0.22微米），保存在4 ° C </li></ul>

isolation of human islets from partially pancreatectomized patients

2型糖尿病胰岛器官捐赠者2有限的2型糖尿病和胰岛故障1胰岛的发病机理的调查而受到阻碍。在这里，我们分享我们的协议从2型糖尿病患者和非糖尿病患者都发生了不同的胰腺疾病（良性或恶性胰腺肿瘤，慢性胰腺炎，胆总管或十二指肠肿瘤）由于部分胰腺切除术的患者获得的人胰腺癌组织分离的胰岛。所有患者参与了他们的同意，这项研究中，也已经由当地伦理委员会的批准。手术标本被立即传送到选定为胰岛隔离柔软和健康出现胰腺组织，保留用于诊断目的的受损组织的病理学家。我们发现，分离出1000多小岛，我们已开始与胰腺组织至少2克。我们的协议还必须是明显扩张的组织注入含酶的媒体时，随后讳言，以帮助消化，增加表面积。 为了扩展我们的协议的适用性，包括大量人力胰腺组织（&gt; 15G）是在偶然情况下，我们使用了Ricordi腔（50毫升）消化组织。在消化过程中，我们手动摇摇Ricordi 厅由试样的强度不同，根据其组织纤维化的程度。然后用一个不连续的聚蔗糖梯度从胰腺组织中分离出来的胰岛细胞。我们注意到，组织沉淀应足够小，密度为1.125克/毫升被均匀悬浮聚蔗糖培养基。隔离后，我们培养了至少48小时的小岛，以促进其功能恢复，下无应力条件下（无需摇动或旋转）5％的CO 2在37 ° C。我们的协议和今后改进的广泛应用，可以使大量从人类糖尿病胰岛适时采收和临床相匹配的非糖尿病患者，极大地推进了2型糖尿病的研究。

我们的协议取得了平均〜500每克胰腺组织胰岛，虽然这极大地之间的准备工作，这主要是由于纤维化和胶原酶活性的差异不同。胰岛纯度&gt; 90％，实现了用双硫腙染色，在组织处理，然后亲手挑选他们隔离后24小时。要确定纯化的胰岛细胞的质量，我们测试了2小时的静态条件下为25毫米葡萄糖刺激胰岛素分泌。我们发现从ECIT胰岛分离和移植中心获得的胰岛的胰岛素分泌相媲美。从部分pancreatectomized的胰腺中分离出的胰岛并不意味着胰岛移植，评估的总的室内环境不被认为是一个关键因素。 更重要的是，我们的方法，使我们分离部分在2005年和2008年5之间的25 pancreatectomized患者胰岛功能的研究。这些胰岛葡萄糖刺激胰岛素分泌和特定的蛋白表达，免疫印迹和免疫组织化学分析。部分pancreatecomized胰腺分离的胰岛细胞也可用于比较基因和蛋白表达谱，超微结构的外观和功能反应的非糖尿病患者和糖尿病胰岛。因为有更多的患者接受部分胰腺切除术相比，有器官捐赠者，我们的协议可能允许强大的统计分析收集到足够的样品和数据。

Watch this Scientific Journal Video about Isolation of Human Islets from Partially Pancreatectomized Patients at JoVE.com

Isolation of Human Islets from Partially Pancreatectomized Patients

为研究2型糖尿病胰岛供应不足。在这里，我们分享我们的协议隔离部分胰腺切除术病人的胰岛。这种方法代表一个独特的场地和2型糖尿病胰岛获得足够数量的非糖尿病患者的临床基础和临床研究相匹配。

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 ...

isolation-of-human-islets-from-partially-pancreatectomized-patients

Research

JoVE Journal

Medicine

15.9K 次观看.  University Hospital Carl Gustav Carus, University of Technology Dresden. 为研究2型糖尿病胰岛供应不足。在这里，我们分享我们的协议隔离部分胰腺切除术病人的胰岛。这种方法代表一个独特的场地和2型糖尿病胰岛获得足够数量的非糖尿病患者的临床基础和临床研究相匹配。

视频: Isolation of Human Islets from Partially Pancreatectomized Patients

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

科研

医学

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

动态视觉测试，以确定和量化视觉损伤与修复继脱髓鞘视神经炎患者

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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, ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

全根主动脉瓣置换主动脉瓣狭窄主动脉异种移植患者小主动脉根

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

足月胎盘胎膜中原发性人蜕细胞的分离

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

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 ...