イリノイ大学シカゴ校だけでなく、膵島細胞リソースセンターNIHの助成金（RFA - RR 05から003）、クリストファー財団、Efroymson財団、およびテラブス財団からの資金によって支え。

1。ファシリティのセットアップ<ol><li>人間の膵島分離には、チームワークを含む時間に敏感な手順であり、それゆえ、4人は、この手順を行うことが必要である。チームは、膵臓の解剖とcannulates外科医によるリードです。 </li><li>膵島分離は、チームはUIC膵島分離施設に入る分を開始します。チームメンバーは、必要なすべての計測機器が、遠心分離機のように、チェックされており、電源が入っていることを確認してください。彼らはまた、すべてのフードとの表面を適切にサニタイズしてください。 </li><li>二人は、カニューレ挿入のテーブル、スラッシュ自販機、消化回路、灌流装置、COBE浄化機、および外科テーブルを設定します。 </li><li>同時に、他の2つのチームのメンバーは、分離時に必要なメディアを準備します。 </li><li>次に、細菌学のチューブ（ボトル、チューブ、フォームおよび袋）、サンプリングチューブ（洗浄とプレ充填）、及び評価のチューブとプレートが用意されています。 </li><li>次のように設定された浄化チューブを準備します：第1回チューブ、空、150 mlでマーク、チューブ2-12は、ヒトアルブミン（洗浄ソリューション）を添加したM199培地200mlで埋め、230 mlでマークされ、そして最終的に1つ50 mlコニカルチューブ洗浄溶液50mlでいっぱい。これらの準備ステップが完了すると我々は、膵臓を作製し、灌流を開始する準備ができました。 </li></ol> 2。膵臓の準備と血流<ol><li>膵臓の準備と灌流を開始するには、演算子は、滅菌テクニックを使用してパッケージから臓器を削除します。外科医は、汚染除去のテーブルに膵臓を転送します。 </li><li>外科医はそれは準備された&quot;調達&quot;細菌学のボトルを埋めるために、それを使用する演算子、にオリジナルの膵臓保存液と手を使用すると、ワン60mlのシリンジを満たします。 </li><li>臓器は、膵島分離（膵臓が調達ま​​たは解剖学的に異常な中に破損している場合、それは膵島分離に使用されていない可能性があります）に適しているかどうかを判断するために目視検査の後、外科医は、コラゲナーゼを準備するためにオペレータが要求されます。この再懸濁の時間が異なる酵素のブランドと多くの間で変化することに注意してください。 </li><li>外科医の準備が整うと、汚染除去のテーブルに（Betadine、Fungizone /セファゾリンおよびHBSS）除染ソリューションを持参し、適切なコンテナにそれらを注ぐ。外科医は、分間除染液の各々に膵臓を洗浄する膵臓の汚染除去を行います。膵臓は、それは重量を測定されるに小さい殺菌した瓶に入れられます。重量は、バッチレコードに入力された後、膵臓は、いくつかの保存液とカニューレのパンに配置されます。 </li><li>外科医は、公開と部分的に主膵管を切開すると、カニューレと膵臓の各半分をカニューレを挿入するでしょう。カニューレは、縫合によって固定されます。膵臓を灌流するには、まず血流のユニットに移動し、流域に酵素液を注ぐ。総灌流時間は約10分、5分では80 mmHgの時、その後5分間で180 mmHgの時です。膵臓は灌流後に膨張した。 </li><li>脂肪組織が慎重に膵臓からトリミングされているカニューレのパンへの灌流膵臓を、移動する。その後10から12の部分に膵臓をカットし、消化のためにRicordiの商工会議所に移す。 </li></ol> 3。膵臓の消化<ol><li> 、消化のステップを開始する空のRicordiのチャンバーに膵臓を転送、Pulmozymeの1つの2.5 mlのバイアルを追加するには、（チャンバーを組織ブロックの大きな塊を避けるために）画面に置かれた、チャンバーを閉じ、システムをいっぱいに。 </li><li> 5分間のソリューションをポンピング開始。ゆっくりと均等に全体の温溶液を分散させるためにチャンバーを揺らし。 5分後、軽く全体の消化の段階のためのチャンバーを振る。 ˚Cで37℃の温度を維持する</li><li>温度が37℃に達するまでゆっくりとチャンバーを揺する、その後チャンバーを振る。サンプルを2分ごとに乗り、無料の島が、スコープの下に発見された場合に検出。バッチ記録の消化テーブル内の膵島評価情報を記録します。そのうちの50％が溶液中に遊離し、分離されたフローティングがあるまで、膵島の割合を監視し続ける。 </li></ol> 4。組織のコレクション<ol><li>かつて島の50％がサンプル中の遊離発見された、消化が回路内にコールドRPMI希釈液を加えることによって停止されます。組織を、500mlコニカルチューブヒトアルブミン30mlで事前入力で収集されます。一度チューブは、スピン清オフ吸引と冷洗浄溶液でペレットを洗浄している。 1分、4˚Cの、1100 RPM（毎分回転数）でコニカルチューブをスピン</li><li>シングル500ミリリットルコニカルに消化された膵臓の組織で中断されたすべてのコレクションチューブを組み合わせる。チューブは洗浄された組織の一杯になると、スピンし、すべての組織が複数回洗浄されるまで、洗浄工程を繰り返します。このチューブをスピンし、単一の250ミリリットルコニカルチューブに組織を移す。 250mlにボリュームをで持ち出すM199洗浄ソリューションでは、HAを補充し、サンプリングの準備。 </li><li>ピペットで1 mlのサンプルを取り、15 mlコニカルチューブ洗浄溶液9 mlをあらかじめ入力して追加します。その後、皿を数える直接グリッドに1ミリリットルからのサンプル、15 mlコニカルチューブゆるやかに転倒によって、本から混ぜる。細胞をカウントし、2500（10x250）の希釈係数を用いて細胞数を計算する。 </li><li> 250ミリリットルコニカルチューブをスピンし、UW（ウィスコンシン大学）は、通常、臓器温存のために使用する溶液の150mlを加える。 UWが追加されたときに、ピペットを用いて細胞懸濁液を混ぜる。 UWの開始時刻を記録し、精製のオペレータに通信する。氷と時折渦巻きにチューブ（s）を残す。今すぐ島をさらに精製することができる。 </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>

分離の結果9,10に影響を与える要因はさまざまです。その中で、膵組織の消化は、重要な役割を果たしている。私たち自身の人間の膵島分離の経験に基づいて、我々は潜在的に膵島の収量と品質に影響を与えうる重要なポイントは、以下の要約。 <ol><li>酵素の様々なタイプが使用されると消化時間が異なっている。 </li><li>早く完全に腺房組織から分離膵島の40〜50％として、消化が希釈溶液...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

1型糖尿病の管理は、年間1,000億ドルで、個人と社会の両方に、厄介です。グルコースモニタリングと新しいインスリン製剤の普及にもかかわらず、多くの個人はまだ壊滅的な二次的合併症を開発する。膵島移植は、糖尿病患者における正常血糖コントロールの近くに復元することができます<sup&gt; 1</sup&gt;、集中的なインスリン療法に関連付けられている重篤な低血糖エピソードのリスクなし。十分な膵島質量を提供することに成功した膵島移植のために重要です。しかし、ドナーの特性、臓器の調達と保存には、分離結果に影響を及ぼす<sup&gt; 2</sup&gt;。イリノイ大学シカゴ校（UIC）で我々は改善された精製勾配で成功した分離プロトコルを開発している<sup&gt; 3</sup&gt;。プログラムは、2004年1月に開始し、300人以上のアイソレーションは、2008年11月までに実施された。膵臓は冷たい保存液（UW、ウィスコンシン州またはHTKの大学、ヒスチジン - トリプトファンケトグルタル酸）に送られ、<sup&gt; 4月7日</sup&gt;膵島分離のためのUICにおける細胞分離の研究室へ。膵島は、もともとRicordiの記載された方法を改変したものですUIC方法を、用いて単離された<em&gt;ら</em&gt;<sup&gt; 8</sup&gt;。簡単に言えば、周囲の組織から膵臓をクリーニングした後、それは酵素溶液（Servaコラゲナーゼ+中性プロテアーゼまたはシグマV酵素）で灌流した。膨張した膵臓は、その後、Ricordiの消化槽に移し、変更、閉鎖循環配管システムに接続し、37℃まで加温した。消化中に、チャンバーを穏やかに振とうした。サンプルは、消化の進行状況を監視するために連続して撮影した。無料の島が顕微鏡下で検出された一度、消化酵素を希釈する循環システムへのコールド（4℃）RPMI希釈液（Mediatech、ハーンドン、バ​​ージニア州）をフラッシュして停止した。収集し、ヒトアルブミンを添加したM199培地で洗浄した後、組織は、事前に精製数をサンプリングし、精製前のUW液でインキュベートした。ヒト膵島の精製と​​文化：精製工程は、第II部で説明される。

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

ヒト膵島の高品質と適切な量を達成することが成功した膵島移植のための著名な前提条件の一つです。このビデオでは、我々は段階的に説明し、人間の膵島を分離するための手順：変更された自動化法を用いて（パートI消化および膵組織のコレクション）。

人間の膵島単離：パートI：膵組織の消化とコレクション

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

human-pancreatic-islet-isolation-part-i-digestion-collection

Research

JoVE Journal

Biology

19.2K ビュー.  University of Illinois, Chicago. ヒト膵島の高品質と適切な量を達成することが成功した膵島移植のための著名な前提条件の一つです。このビデオでは、我々は段階的に説明し、人間の膵島を分離するための手順：変更された自動化法を用いて（パートI消化および膵組織のコレクション）。

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

Murine Pancreatic Islet Isolation

マウス膵島単離

生物学

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.

人間の膵島単離：パートII：ヒト膵島の精製と​​文化

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.

ヒト膵臓癌異種移植片由来の幹細胞の分離

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.

膵島質量、サイズ分布とアーキテクチャのコンピュータ支援大規模可視化と定量化

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.

マウス膵島の単離と細胞内cAMP測定法

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.

マウスプライマリ膵臓腺房細胞の単離と培養

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.

マウスからの膵島分離のための簡単な高効率プロトコル

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.

パラフィン セクションの埋め込み膵島

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.

マウス膵島細胞の亜集団におけるカルシウムダイナミクスのイメージング

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

ヒト多能性幹細胞の膵臓β細胞への分化

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.

著者スポットライト:多能性幹細胞からのβ細胞分化における進歩と課題

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.