بدعم من التمويل من جامعة إلينوي في شيكاغو ، وكذلك جزيرة الخلية مركز موارد منحة المعاهد الوطنية للصحة (RFA - RR 05-003) ، ومؤسسة كريستوفر ، ومؤسسة Efroymson ، ومؤسسة Tellabs.

1. مرفق الإعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الإنسان عزلة الجزيرة هو إجراء الوقت الحساسة التي تنطوي على العمل الجماعي ، وبالتالي ، هناك حاجة إلى أربعة أشخاص لإجراء هذا الإجراء. ويقوم الفريق بقيادة الجراح الذي يشرح وcannulates البنكرياس. </li><li style=";text-align:right;direction:rtl"> عزلة الجزيرة تبدأ في اللحظة التي يدخل فريق جزيرة العزلة UIC المرفق. أعضاء فريق التأكد من أن جميع الأجهزة اللازمة ، مثل أجهزة الطرد المركزي ، ويتم التحقق قيد التشغيل. أيضا التأكد من أنهم لتعقيم جميع اغطية والسطوح. </li><li style=";text-align:right;direction:rtl"> سوف شخصين إعداد جدول إقناء ؛ إدخال القنية ، وآلة للتبذير ، والدوائر الهضم ، وحدة التروية ، كوبي آلة التنقية ، والجداول الجراحية. </li><li style=";text-align:right;direction:rtl"> في نفس الوقت ، سوف اثنين من أعضاء الفريق الآخر إعداد وسائل الاعلام ان هناك حاجة خلال العزلة. </li><li style=";text-align:right;direction:rtl"> المقبل ، وتعد أنابيب البكتريولوجيا (الزجاجات والأنابيب وشكل وكيس) ، وأنابيب أخذ العينات (ما قبل مليئة يغسل) ، وأنابيب وألواح التقييم. </li><li style=";text-align:right;direction:rtl"> إعداد أنبوب تنقية النحو التالي : 1 أنبوب فارغة وضعت في 150 مل ، وأنابيب مملوءة 2-12 200 مل من وسائل الاعلام M199 تستكمل مع الإنسان (محلول الغسيل) الألبومين وضعت في 230 مل ، وأخيرا أنبوب واحد مل 50 المخروطية مليئة 50 مل من محلول الغسيل. بمجرد إعداد هذه الخطوات هي كاملة ونحن على استعداد لبدء إعداد وperfusing البنكرياس. </li></ol> 2. إعداد البنكرياس ونضح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لبدء الإعداد ونضح من البنكرياس ، وهو عامل من يزيل الجهاز باستخدام تقنية التغليف المعقم. وسوف يقوم الجراح نقل البنكرياس إلى طاولة التطهير. </li><li style=";text-align:right;direction:rtl"> وسوف يقوم الجراح ملء أحد حقنة 60 مل مع الحل البنكرياس الحفاظ الأصلي وتسليمه لأحد المشغلين ، الذي سيتم استخدامه لملء الزجاجات المعدة &quot;المشتريات&quot; الجراثيم. </li><li style=";text-align:right;direction:rtl"> بعد الفحص البصري لتحديد ما إذا كان الجهاز هو مناسب لعزل الجزيرة (في حالة تلف البنكرياس خلال الشراء أو غير طبيعية تشريحيا ، قد لا يمكن استخدامه لعزل الجزيرة) ، سوف الجراح يطلب من عامل لإعداد كولاجيناز. نتذكر أن ذلك الوقت إعادة تعليق يتراوح بين الماركات المختلفة ، والكثير الانزيم. </li><li style=";text-align:right;direction:rtl"> عندما الجراح مستعد ، وجلب الحلول التطهير (Betadine ، Fungizone / سيفازولين وHBSS) الى طاولة المفاوضات من أجل إزالة التلوث ، ومنهم في حاويات مناسبة. الجراح ينفذ التطهير البنكرياس الغسيل لمدة دقيقة والبنكرياس في كل من الحل إزالة التلوث. ثم يوضع البنكرياس في جرة صغيرة معقمة الذي توزن به. بعد إدخال وزن في السجل الدفعي ، ويوضع البنكرياس في عموم إقناء ؛ إدخال القنية مع بعض الحلول للحفظ. </li><li style=";text-align:right;direction:rtl"> وكشف الجراح وجزئيا شق القناة الرئيسية ويقني ؛ يدخل القنية كل شوط من البنكرياس مع قنية. وسيتم تأمين قنية بواسطة خياطة. ليروي البنكرياس ، حرك لأول مرة إلى وحدة ونضح من أجل حل الانزيم في الحوض. إجمالي الوقت نضح حوالي 10 دقيقة ، بمعدل 80 ملم زئبقي لمدة 5 دقائق ، ثم عند 180 مم زئبق لمدة 5 دقائق. منتفخة البنكرياس بعد نضح. </li><li style=";text-align:right;direction:rtl"> نقل البنكرياس perfused لعموم إقناء ؛ إدخال القنية ، حيث يتم قطع الأنسجة الدهنية من البنكرياس بعناية. ثم قص البنكرياس في 10-12 القطع ونقلها إلى غرفة Ricordi للهضم. </li></ol> 3. البنكرياس الهضم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لبدء خطوة عملية الهضم ، ونقل البنكرياس إلى الدائرة Ricordi فارغة ، إضافة واحد مل فيال 2.5 من Pulmozyme ، وطرح على الشاشة (تجنب جزءا كبيرا من كتلة النسيج غرفة) ، على مقربة من القاعة ، وتملأ النظام. </li><li style=";text-align:right;direction:rtl"> بدء ضخ المحلول لمدة 5 دقائق. صخرة بلطف الغرفة لتفريق بالتساوي حل الدافئة طوال الوقت. بعد خمس دقائق ، ويهز بلطف الغرفة للمرحلة الهضم برمتها. المحافظة على درجة حرارة 37 ˚ C. </li><li style=";text-align:right;direction:rtl"> موسيقى الروك في غرفة بلطف حتى تصل درجة الحرارة الى 37 درجة مئوية ، ثم يهز الغرفة. أخذ عينة كل دقيقتين وكشف إذا تم العثور على الجزر حرة تحت نطاقها. تسجيل معلومات التقييم جزيرة في الجدول الهضم من المحضر دفعة واحدة. تواصل رصد نسبة الجزر حتى هناك 50 ٪ منهم من التعويم ومعزولة في الحل. </li></ol> 4. جمع الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مرة واحدة تم العثور على 50 ٪ من الجزر الحرة في العينات ، وتوقف عملية الهضم وذلك بإضافة الباردة حل تخفيف RPMI في الدائرة. ويتم جمع الأنسجة في أنابيب مخروطية 500 مل قبل مليئة الزلال 30 مل الإنسان. حالما يتم نسج أنابيب شفط قبالة طاف بيليه وتغسل بمحلول الغسيل الباردة. تدور الأنابيب المخروطية في 1100 دورة في الدقيقة (التناوب في الدقيقة) لمدة 1 دقيقة ، 4 ˚ جيم </li><li style=";text-align:right;direction:rtl"> الجمع بين جميع أنابيب جمع العالقة مع أنسجة البنكرياس هضمها في المخروطية واحد 500 مل. عندما أنبوب مليء الأنسجة غسلها ، وتدور وتكرار عملية الغسيل حتى تم غسلها جميع أنسجة عدة مرات. تدور هذه الأنابيب ونقل الأنسجة في أنبوب واحد 250 مل المخروطية. جعل حجم ما يصل الى 250 مل معM199 حل الغسيل تستكمل مع HA والاستعداد لأخذ العينات. </li><li style=";text-align:right;direction:rtl"> أخذ عينة 1 مل مع ماصة وإضافة إلى أنبوب 15 مل المخروطية قبل مليئة 9 مل من محلول الغسيل. ثم مزيج من قلب بلطف أنبوب مخروطي 15 مل و من هذا ، من أصل عينة 1ml مباشرة في صحن شبكة العد. عدد الخلايا ، وحساب عدد الخلايا باستخدام عامل إضعاف 2500 (10x250). </li><li style=";text-align:right;direction:rtl"> تدور في أنبوب 250 مل المخروطية وإضافة 150 مل من UW (جامعة ويسكونسن) التي تستخدم عادة الحل للحفاظ على الجهاز. عند إضافة UW ، مزيج تعليق الخلية بدقة باستخدام ماصة. سجل والتواصل للمشغل لتنقية وقت البدء في UW. ترك الأنبوب (ق) على الجليد ودوامة أحيانا. الآن يمكن تنقيته من جزر أخرى. </li></ol>

<ol>
	<li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Nano, R., 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=Islet+isolation+for+allotransplantation:+variables+associated+with+successful+islet+yield+and+graft+function.">Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.</a> Diabetologia. 48, 906-912 (2005).</li><li>Barbaro, B., 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=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84, 1200-1203 (2007).</li><li>Lakey, J. R., Rajotte, R. V., Warnock, G. L., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pancreas+preservation+prior+to+islet+isolation.+Cold+ischemic+tolerance.">Human pancreas preservation prior to islet isolation. Cold ischemic tolerance.</a> Transplantation. 59, 689-694 (1995).</li><li>Fridell, J. A., 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=Comparison+of+histidine-tryptophan-ketoglutarate+solution+and+University+of+Wisconsin+solution+for+organ+preservation+in+clinical+pancreas+transplantation.">Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation.</a> Transplantation. 77, 1304-1306 (2004).</li><li>Potdar, S., 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=Initial+experience+using+histidine-tryptophan-ketoglutarate+solution+in+clinical+pancreas+transplantation.">Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation.</a> Clin Transplant. 18, 661-665 (2004).</li><li>Salehi, P., 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=Human+islet+isolation+outcomes+from+pancreata+preserved+with+Histidine-Tryptophan+Ketoglutarate+versus+University+of+Wisconsin+solution.">Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution.</a> Transplantation. 82, 983-985 (2006).</li><li>Ricordi, C., Lacy, P. E., 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+islet+isolation+from+human+pancreas.">Automated islet isolation from human pancreas.</a> Diabetes. 38, 140-142 (1989).</li><li>Hanley, S. C., Paraskevas, S., Rosenberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+and+isolation+variables+predicting+human+islet+isolation+success.">Donor and isolation variables predicting human islet isolation success.</a> Transplantation. 85, 950-955 (2008).</li><li>Toso, C., 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=Factors+affecting+human+islet+of+Langerhans+isolation+yields.">Factors affecting human islet of Langerhans isolation yields.</a> Transplant Proc. 34, 826-827 (2002).</li></ol>

وهناك مجموعة متنوعة من العوامل التي تؤثر على نتائج العزلة 9،10. فيما بينها ، والهضم للانسجة البنكرياس يلعب دورا بارزا. بناء على منطقتنا الإنسان جزيرة تجربة العزلة ، لخص لنا النقاط التالية الحاسمة ، التي يمكن أن تؤثر المحتمل أن تسفر جزيرة والجودة. <ol style=";text-align:right;directi...

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

human pancreatic islet isolation: part i: digestion and collection of pancreatic tissue

إدارة مرض السكري نوع 1 هو مرهقة ، سواء على الفرد والمجتمع ، وتكلف أكثر من 100 مليار دولار سنويا. على الرغم من الاستخدام الواسع النطاق للرصد الغلوكوز والانسولين صياغات جديدة ، وتطوير العديد من الأفراد لا تزال مضاعفات ثانوية مدمرة. ويمكن زرع البنكرياس جزيرة بالقرب من استعادة السيطرة على مستويات الجلوكوز الطبيعي في مرضى السكري<sup&gt; 1</sup&gt; ، دون التعرض لخطر نوبات سكر الدم الخطيرة التي ترتبط مع العلاج المكثف بالأنسولين. توفير ما يكفي من الكتلة جزيرة المهم للجزيرة زرع ناجحة. ومع ذلك ، سمة من الجهات المانحة ، والمشتريات ، والحفاظ على الجهاز تؤثر على النتيجة عزلة<sup&gt; 2</sup&gt;. في جامعة إلينوي في شيكاغو (UIC) وضعنا بروتوكولا العزلة ناجحة مع التدرج تحسين تنقية<sup&gt; 3</sup&gt;. بدأ البرنامج في يناير 2004 ، وأجريت أكثر من 300 من العزلة حتى نوفمبر 2008. تم إرسال بنكرياسات في حلول الحفاظ الباردة (UW ، جامعة ويسكونسن أو HTK ، الحامض الأميني تريبتوفان كيتوغلوتارات)<sup&gt; 4-7</sup&gt; لخلية مختبر العزل في المحاكم الاسلامية لعزل الجزيرة. وقد تم عزل جزيرات البنكرياس باستخدام الأسلوب UIC ، الذي هو نسخة معدلة من الطريقة الموصوفة في الأصل من قبل Ricordi<em&gt; وآخرون</em&gt;<sup&gt; 8</sup&gt;. لفترة وجيزة ، وبعد تنظيف البنكرياس من الأنسجة المحيطة بها ، وكان ذلك مع حل perfused انزيم (Serva كولاجيناز الانزيم + V أو متعادلة البروتياز سيغما). ثم نقل البنكرياس منتفخة إلى غرفة Ricordi الهضم ، متصلة ، أغلقت تعديل نظام التداول الأنابيب ، وارتفعت درجة حرارة تصل إلى 37 درجة مئوية. خلال عملية الهضم ، واهتزت الغرفة بلطف. وقد أخذت عينات باستمرار لرصد التقدم الهضم. مرة واحدة تم الكشف عن الجزر حرة تحت المجهر ، وتوقفت عملية الهضم من خلال تنظيف الباردة (4 درجات مئوية) حل تخفيف RPMI (Mediatech ، هيرندون ، فرجينيا) في نظام التداول لتخفيف الانزيم. بعد أن تم جمعها وغسلها وسائل الاعلام في M199 تستكمل مع الزلال البشري ، وأخذت عينات الأنسجة لتنقية ما قبل العد وحضنت مع الحل UW قبل التنقية. ويمكن وصف عملية تنقية في الجزء الثاني : تنقية الثقافة والجزر الإنسان.

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue at JoVE.com

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

تحقيق الجودة العالية وكمية مناسبة من الجزر الإنسان هي واحدة من أبرز الشروط لنجاح زرع جزيرة. في هذا الفيديو ، ونحن تصف خطوة بخطوة لإجراءات العزل جزيرة البنكرياس الإنسان (الجزء الأول : الهضم ، وجمع من نسيج البنكرياس) باستخدام طريقة تعديل الآلي.

الإنسان عزل جزيرة البنكرياس : الجزء الأول : الهضم ومجموعة من الأنسجة البنكرياس

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

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Biology

19.2K Views.  University of Illinois, Chicago. تحقيق الجودة العالية وكمية مناسبة من الجزر الإنسان هي واحدة من أبرز الشروط لنجاح زرع جزيرة. في هذا الفيديو ، ونحن تصف خطوة بخطوة لإجراءات العزل جزيرة البنكرياس الإنسان (الجزء الأول : الهضم ، وجمع من نسيج البنكرياس) باستخدام طريقة تعديل الآلي.

Video: Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Murine Pancreatic Islet Isolation

فأري عزل جزيرة البنكرياس

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

الإنسان عزل جزيرة البنكرياس : الجزء الثاني : تنقية الثقافة والإنسان من الفشوت

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

عزل الخلايا الجذعية من الإنسان Xenografts سرطان البنكرياس

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

بمساعدة الحاسوب على نطاق واسع التصور النوعي والكمي لقداس جزيرة البنكرياس والتوزيع الحجم والعمارة

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

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

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

وهناك طريقة للماوس البنكرياس جزيرة العزلة وبين الخلايا المخيم تقدير

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

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

العزلة والثقافة من الماوس الأساسي خلايا البنكرياس عنيبية

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

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

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

بروتوكول بسيط عالي الكفاءة لعزل الجزيرة البنكرياسية عن الفئران

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

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

جزيرة البنكرياس التضمين لمقاطع البارافين

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

تصوير ديناميات الكالسيوم في السكان الفرعيين من خلايا الفئران جزيرة البنكرياس

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

تمييز الخلايا الجذعية البشرية متعددة القدرات إلى خلايا β البنكرياس

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

تسليط الضوء على المؤلف: التطورات والتحديات في تمايز الخلايا β من الخلايا الجذعية متعددة القدرات

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.