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人类胰岛的分离：第一部分：收集和胰腺组织消化

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Department of Surgery, University of Illinois, Chicago

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Cite this Article: 人类胰岛的分离：第一部分：收集和胰腺组织消化

Qi, M., Barbaro, B., Wang, S., Wang, Y., Hansen, M., Oberholzer, J. Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue. J. Vis. Exp. (27), e1125, doi:10.3791/1125 (2009).

Abstract: 人类胰岛的分离：第一部分：收集和胰腺组织消化

1型糖尿病的管理负担，既要对个人和社会，每年耗资超过100亿美元。尽管血糖监测和新的胰岛素制剂的广泛使用，许多人仍然发展毁灭性的继发性并发症。胰岛移植可以恢复接近正常的糖尿病患者血糖控制

Protocol: 人类胰岛的分离：第一部分：收集和胰腺组织消化

1。设备安装

  1. 人胰岛分离是一个时间敏感的程序，涉及团队精神，因此，四个人都需要进行此步骤。该小组是导致受术者，剖析和cannulates胰腺。
  2. 胰岛隔离分钟开始，球队进入UIC的胰岛隔离设施。小组成员，确保一切必要的检测仪器，如离心机，检查并打开。他们还确保消毒所有的油烟机和表面。
  3. 两个人将成立插管表，雪泥机，消化电路，灌注单元，COBE净化机，手术表。
  4. 同时，两个团队的其他成员将作好准备，在隔离期间所需的媒体。
  5. 接下来，细菌学管（瓶，管，形式和袋），采样管（洗预填充），并评估管和板的准备。
  6. 准备净化管设置如下：第一空管和150毫升的标记，管2-12充满M199媒体补充人血白蛋白（洗涤液）200毫升和230毫升标记，终于有50 ml锥形管充满了洗涤液50毫升。一旦这些准备步骤完成后，我们就可以开始准备和灌流胰腺。

2。胰腺的准备和灌注

  1. 要开始准备和胰腺灌注，操作员将删除从使用无菌包装的器官。胰腺外科医生将转移到去污表。
  2. 医生会填写一个与原来的胰腺保存液和操作员，他们将使用它来填充编写的“采购”细菌学瓶60毫升注射器。
  3. 一次视力检查，以确定器官是否适合胰岛隔离（如果胰腺的损坏在采购或解剖结构异常，它可能不能使用胰岛隔离）后，医生会要求运营商准备胶原酶。请记住，再悬浮的时间，不同的酶的品牌和地段之间的不同。
  4. 当医生准备好了，带来的净化解决方案（优碘，Fungizone /头孢唑啉和HBSS）的去污表，倒入适当的容器内。外科医生进行一分钟的洗涤去污解决方案中的每个胰腺的胰腺去污。胰腺，然后放置在一个小无菌罐，它会权衡。胰腺后的重量是输入到批处理记录，是摆在插管泛一些保存液。
  5. 医生会揭露和部分切割主要的胆管和cannulate胰腺与套管各占一半。将套管固定缝合。灌注胰腺，首先将其移动到灌注单元和酶液倒入盆地。共有灌注时间约10分钟，5分钟在80毫米汞柱，然后在5分钟的180毫米汞柱。胰腺扩张后灌注。
  6. 移动插管泛灌注胰腺，脂肪组织是从胰腺精心修剪。然后切成10-12块胰腺的消化和转移Ricordi商会。

3。胰腺消化

  1. 要开始消化步骤，胰腺转移到空Ricordi厅，添加2.5毫升Pulmozyme的小瓶，把屏幕上（避免大块组织块室），接近香港总商会，并填写系统。
  2. 开始抽5分钟的解决方案。轻轻摇动室，均匀地分散温暖的解决方案，整个。五分钟后，轻轻摇动整个消化阶段会议厅。温度保持在37˚C。
  3. 轻轻晃动室，直至温度达到37℃，然后摇动室。每两分钟取一次样和检测，如果发现免费胰岛范围。记录在批记录的消化表中的胰岛评价信息。继续监测胰岛的百分比，直到有解决方案中的其中50％的浮动自由和孤立。

4。收集组织

  1. 一旦发现50％的胰岛的样品中，消化停止在电路中加入冷的RPMI稀释液。组织收集了500毫升与30毫升的人血白蛋白预充的锥形管。一旦纺管，吸关闭上清，用冷水洗涤液洗沉淀。在1100 RPM（每分钟旋转）旋转锥管为1分钟，4˚C。
  2. 消化胰腺组织成一个单一的500毫升锥形暂停的，把所有收集管。当管洗净组织，自旋和重复洗涤过程中，直到所有的组织已经多次洗涤。旋转管和组织转移到一个单一的250毫升的锥形管。把音量高达250毫升M199洗涤液补充与医管局及准备采样。
  3. 用移液器取1毫升样品，并添加到15毫升锥形管与洗涤液9毫升预充。然后轻轻颠倒15毫升的锥形管，从这个混合出1毫升的样品直接计数盘成一个网格。计数细胞，并计算细胞数，使用稀释倍数2500（10x250）。
  4. 旋转250毫升的锥形管，加入150毫升威斯康星大学（美国威斯康辛大学）解决方案，通常用于器官保存。当威斯康星添加，彻底混合细胞悬液，用吸管。记录和沟通，以净化运营商在威斯康星大学的开始时间。留在冰和漩涡偶尔管（S）。现在的胰岛细胞能进一步得到净化。

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Discussion: 人类胰岛的分离：第一部分：收集和胰腺组织消化

有多种因素影响隔离的结果9,10。其中，消化胰腺组织中起着突出的作用。基于我们自己的人胰岛隔离的经验，我们总结了以下关键点，这有可能影响胰岛的产量和质量。

  1. 时利用不同类型的酶，消化时间是不同的。
  2. 只要完全从胰腺组织中分离胰岛的40-50％，消化应立即停止加入稀释液，冷却下来的组织。
  3. 柔和的组织处理，收集，洗涤，和混合。
  4. 收集组织和UW液的混合物，应在冰上放置至少30分钟，以获得较好的净化效果。

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Disclosures: 人类胰岛的分离：第一部分：收集和胰腺组织消化

Acknowledgements: 人类胰岛的分离：第一部分：收集和胰腺组织消化

在伊利诺伊大学芝加哥分校以及胰岛细​​胞资源中心美国国立卫生研究院授予（05-003 RFA - RR），克里斯托弗基金会，该Efroymson基金会，和泰乐基金会的资金支持。

Materials: 人类胰岛的分离：第一部分：收集和胰腺组织消化

Name Type Company Catalog Number Comments
Ice Slush Machine equipment Taylor Company K4036546 make sterile ice
Centrifuge equipment Beckman Coulter Inc. 424803
MZ6 Inverted Microscopy TLBD4.1 equipment Leica Microsystems 4103
Cell Culture Dish with 2 mm Grid equipment Nalge Nunc international 174926 islet counting
Digital Water Bath EX7 equipment NesLab 105361058
Thermometer equipment IBI:IR 60444
MonoBloc Balance AB 104-S equipment Mettler Toledo 1119430864 measure pancreas weight
MasterFlex II Speed Controller equipment Cole-Parmer J00006445 adjust flow speed during degestion
Digital Sight DS L1 equipment Nikon Instruments 217267
Perfusion Unit equipment Custom Made n/a
Cooler for perfusion unit equipment Fisher Scientific 3013P
Ricordi Chamber equipment University of Miami n/a
RPMI dilution solution reagent Mediatech, Inc. 99783024
University of Wisconcin (UW) solution reagent DURAMED 1000-46-06
Hank’s Buffered Salt Solution (HBSS) reagent Mediatech, Inc. 99-597-CM
25% Human Albumin reagent GRIFOLS NDC 61953-0002-2
M199 media (wash solution) reagent Mediatech, Inc. 99-784-CM
Collagenase NB1 GMP grade reagent SERVA Electrophoresis N0002937
Neutral Protease NB1 GMP grade reagent SERVA Electrophoresis N0002936
Betadine reagent Cardinal Health 29906-004
Cefazolin reagent West-Ward Pharmaceutical NDC 0143-9924-90
Dithizone reagent Sigma-Aldrich D5130

References: 人类胰岛的分离：第一部分：收集和胰腺组织消化

  1. Shapiro, A.M., et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238 (2000).
  2. Nano, R., et al. Islet isolation for allotransplantation: variables associated with successful islet yield and graft function. Diabetologia 48, 906-912 (2005).
  3. Barbaro, B., et al. Improved human pancreatic islet purification with the refined UIC-UB density gradient. Transplantation 84, 1200-1203 (2007).
  4. Lakey, J.R., Rajotte, R.V., Warnock, G.L. & Kneteman, N.M. Human pancreas preservation prior to islet isolation. Cold ischemic tolerance. Transplantation 59, 689-694 (1995).
  5. Fridell, J.A., et al. Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation. Transplantation 77, 1304-1306 (2004).
  6. Potdar, S., et al. Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation. Clin Transplant 18, 661-665 (2004).
  7. Salehi, P., et al. Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution. Transplantation 82, 983-985 (2006).
  8. Ricordi, C., Lacy, P.E. & Scharp, D.W. Automated islet isolation from human pancreas. Diabetes 38 Suppl 1, 140-142 (1989).
  9. Hanley, S.C., Paraskevas, S. & Rosenberg, L. Donor and isolation variables predicting human islet isolation success. Transplantation 85, 950-955 (2008).
  10. Toso, C., et al. Factors affecting human islet of Langerhans isolation yields. Transplant Proc 34, 826-827. (2002).

Ask the Author: 人类胰岛的分离：第一部分：收集和胰腺组织消化

5 Comments

Nice video, Barbara. I learned a lot.

Tatsuya from Edmonton

1

Reply

Posted by: Tatsuya K.May 28, 2009, 7:34 PM

Thank you tatsuya

1.1

Reply

Posted by: BarbaraMay 28, 2009, 7:45 PM

i am from taiwan, a transplant surgeon. it is very impressive to see this video and really learn a lot! appreciate your kindly share!

2

Reply

Posted by: hsinlin tsaiJuly 14, 2009, 12:28 PM

if you want to learn, please let us know. Really appreciate your comments. Yong

2.1

Reply

Posted by: YongJuly 14, 2009, 1:02 PM

I also think this video is very helpful. I just joined as a research assistant at an islet transplant lab in Canada. I just wish I could download the movie to view it more often.

3

Reply

Posted by: Marco G.October 31, 2010, 7:20 PM

Thanks for your comments. You can subscribe and download the movie from the web.

3.1

Reply

Posted by: MeirigengNovember 1, 2010, 10:07 AM

Great video! Definitely very helpful in visualizing the protocol. Are you able to provide any additional information on the perfusion unit set up?

4

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Posted by: Brooke L.December 12, 2012, 11:50 AM

Hello Brooke L, thank you so much for sending this massage and being interested in our human islet isolation protocol. I can certainly provide more information and discuss the perfusion unit set up. Please send us an email about what exactly you want to know. We are happy to give more explanations. Thanks

5

Reply

Posted by: Meirigeng Q.December 12, 2012, 2:44 PM

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