Name: effective isolation of functional islets from neonatal mouse pancreas
Uploaded: 2017-01-06T11:30:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

動物の使用は、プロトコルM /区のためのヴァンダービルト施設内動物管理使用委員会によって承認された181分の11で指定された手順に従います。 CD1またはCBA / BL6マウスは、市販業者から購入し、新生児マウスを得るために、ヴァンダービルトの動物施設で交配しました。 1.マウスの作製、ストックソリューション、および機器<ol><li>マウスのクロスの場合：マウスのクロスを設定し、実験計画を支援するために差し込む日付を記録します。 CD1マウスは、通常、交配後19日目の周りに出産します。使用CD1またはCBA / BL6は、それぞれ、CD1またはCBA / BL6新生児を得ることが横切ります。 2ライン間の交雑は、CD1雌及びCBA / BL6雄を利用します。 </li><li>コラゲナーゼ在庫について：高精密天秤で200mgのコラゲナーゼタイプIVの重量を量ります。 50 mL遠心管に移します。カルシウムと40 mLのHBSSに溶かし2+ / Mgの2+（それぞれ1.26および0.5 mMのは、）を5mg / mLのストック溶液を作製しました。 <ol><li>へのコラゲナーゼを許可します穏やかに振盪しながら〜30分間溶解します。少なくとも1年間有効のままになりストックとして-20℃で凍結させた溶液を、アリコートとキープ。 </li></ol></li><li>解剖および島ピッキングのためのピンセット、はさみ、およびP20のヒントのいくつかのペアを滅菌するオートクレーブ。 </li><li> 1 Mグルコース在庫について：グルコースを溶解するために50mlのRPMI 1640培地を追加し、50 mL遠心管に9グラムのグルコースを量ります。使用するまで4℃に保ち、ない6カ月以上。 </li></ol> 2.ワーキングソリューション注：膵島単離の日は、以下の試薬を準備します。 <ol><li>コラゲナーゼIV型作業溶液の場合：解凍し、氷上でコラゲナーゼの株式を希釈します。各1.7 mLのマイクロチューブに0.15 mLのストックを分注します。 Ca 2+ / Mgの2+で1.35 mLのHBSSを追加します。チューブを5回反転します。氷上で15分間のままにしておきます。 <ol><li>微量で3分間、高速で回転。上清を新しいチューブに移します。使用するまで氷上に残します。不溶性の破片でチューブを捨てます。 </li></ol></li><li>完全RPMI 1640培地の場合：500 mLのRPMI 1640培地に2.5 mLの1 Mグルコース、FBS不活化50 mLの熱、および5 mLの100倍ペン連鎖球菌の株式を追加します。使用するまで氷上におきます。 </li></ol> 3.膵臓の単離と消化<ol><li>承認されたプロトコルに従って断頭続いイソフルランと新生児を、安楽死させます。 </li><li>その腹を上に向けてマウスを置きます。殺菌のための70％エタノールでスプレーします。 </li><li>ピンセットで腹の皮膚を持ち上げ、陰部から胸郭に、ハサミで正中線に沿って縦方向に皮膚や筋肉層を切ります。これは、すべての内臓を露出させるために腹腔を開きます。 </li><li>ピンセットで100センチの皿にすべての内臓を転送します。胃と脾臓と十二指腸11にしがみつい腹部分にしがみつい背部分で散乱臓器である膵臓を、見つけます。 </李&gt; <li>臓器を沈めるために皿にHBSSを追加します。溶液中では、膵臓組織はもはや十二指腸、胃、または脾臓にしがみつくません。それは、その典型的な白い色の解剖範囲の下で容易に認識可能です。 HBSSで新しい60ミリメートル皿に離れて周囲の組織と転送から膵臓組織を剥離するためにピンセットを使用してください。 </li><li>任意のディメンション間5ミリメートルより小さな部分にハサミで各膵臓をカットします。 1.5 mLのマイクロ遠心チューブに移します。 5膵臓（P7より若い）まで1チューブ内で消化することができます。 P8-P16マウスの場合、膵臓あたり1チューブを使用しています。 </li><li>各チューブに1 mLのコラゲナーゼ作業溶液（それぞれP1-P7の膵臓用0.2 mLの各P8-P16膵臓のために0.5 mLのを使用してください。） - 0.5を追加します。最大15分間37℃のインキュベーター内でチューブを残します。チューブを2回毎分を反転。 </li><li> 〜5分後、膵臓組織の消化状況を監視するためにチューブをタップします。膵臓のほとんどまで消化プロセスを続行組織は、直径&lt;2ミリメートルで、断片化された細胞塊として表示されます。溶解物を、通常の腺房細胞の自己消化によるものである、曇って表示されます。 </li></ol> 4.溶解物の洗濯<ol><li>微量で10秒間500×gで溶解液をスピン。 P1,000ピペットマンで上澄み層を除去。上清は濁ったが、細胞クラスターを欠いて表示されます。すぐに起因するいくつかの粘着性のDNAの存在に下部の組織片を離れて吸引し得る、吸引を使用しないでください。 </li><li> 1 mLのRPMI 1640完全培地を追加します。断片化した溶解物を再懸濁するために静かにチューブをタップします。アップ裏返し、10回ダウン。繰り返し手順4.1。 </li><li>繰り返しステップ4.2をさらに2回または上清が透明になるまで。 1 mLの完全RPMI 1640培地で洗浄した膵臓の断片を再サスペンド。 </li></ol> 5.膵島単離注：膵臓の小さな番号については、直接手で摘み、以下のように（5.1）は、膵島ISOLのために使用することができますエーション。膵臓の多数のために（&gt; 6）、5.2に概説された方法が好ましいです。 <ol><li>直接手でピッキング： <ol><li> 60ミリメートル皿にステップ4.3からの溶解物を転送します。 5 mLの完全RPMI 1640培地を追加します。解剖顕微鏡下で持参してください。 </li><li>顕微鏡での明視野照明を設定します。最大電力の約50％の光量を調整します。この状態で、島は腺房細胞のような暗い不規則な形のクラスターのに対し（ 図1A）、など少しピンクのクラスタを表示されます。 （対物レンズと接眼のパワーを組み合わせる）〜50Xの倍率は、小島とピッキングに対して同時に開くピペットチップを可視化する最良の方法を提供することをお勧めします。 </li><li>腺房クラスタを避けながら、P20のピペットチップで膵島をピックアップ。 RPMI完全培地（ 図1B）を持つ新しい60ミリメートル皿に小島-濃縮された画分を分注します。 </li><li>二回以上手でピッキングプロセスを繰り返します。新しい60で純粋な島を残します4 mLの完全RPMI培地（ 図1C）とmmディッシュ。 </li></ol></li><li>勾配遠心分離： <ol><li> 1.077グラム/ mlの密度で2mLのポリスクロースおよびジアトリゾ酸ナトリウムがプリロードされた15 mL遠心チューブを準備します。使用するまで氷上でチューブを残します。 </li><li>パスツールピペットでポリスクロースおよびナトリウムジアトリゾ液の上部にステップ4.3からサスペンションを転送します。底層を乱さないでください。最大10の組織が新生または2 P17膵臓を各15mLの管に装填することができます。 </li><li> 4℃で15分間、スイングローターで〜600×gでチューブをスピン。ブレーキオフの設定を使用します。 </li><li>遠心分離した後、新しい15mLのチューブに（理想的な動作条件の下で、島は遠心分離後ポリスクロースと文化の媒体間の相間に配置されますことに注意してください）メディアと相間層を転送します。 10 mLのメディアを追加し、穏やかに、しかし完全に混合します。 </li><li>膵島濃縮画分をスピンダウン5分間、300×gで。洗浄培地を除去します。完全RPMI培地で洗浄をもう一度繰り返します。主に膵島およびダクト（ 図1D）が含まれている 4 mLのメディアへの再懸濁ペレット。 </li><li>セクション5.1に概説された膵島を手で選びます。 </li></ol></li></ol>単離された膵島6. GSISアッセイ<ol><li> 37℃の組織培養インキュベーター中で2時間、完全RPMI培地中でステップ5.1.4から膵島を培養します。 </li><li>解剖顕微鏡下でRPMIを削除するには、ピペットを使用してください。プレート3に2.8 mMグルコースと3 mLのKRBのソリューションを追加します。膵島を洗浄するために10回渦巻。 KRBソリューションを削除します。 <ol><li>一度洗浄工程を繰り返します。インキュベーター中で1時間、37℃で3 mLのKRB溶液で島をインキュベートします。 </li></ol></li><li> 12ウェルプレートの各ウェルにアリコート1mLのKRB。インキュベーター中で37℃にKRBストックとプレートを事前に温めます。 </li><li> 6.2のように予熱したKRBで二回の膵島を洗ってください。トランスFER予熱したプレートの各ウェルに10の島。 37℃のインキュベーター中でインキュベートします。 </li><li> 45分後、50μLの上清を撤回。これは、基礎グルコースの下で分泌されるインスリンのソリューションが含まれています。 </li><li> 32μL500 mMグルコースを追加します。溶液を混合するためにプレートを20回渦巻。 37℃のインキュベーター中でインキュベートします。プレートを5回ごとに5分を渦巻。 </li><li> 45分後、50μLの上清を撤回。これは、高グルコース下で分泌されるインスリンです。 </li><li> 1.5 mLのチューブに、すべての膵島を転送します。 30分間-20℃で凍結します。 5分間、室温で解凍します。凍結融解処理を繰り返します。 </li><li> 20mMのHClで0.5 mLの70％エタノールを追加します。一晩おきます。これは、ランゲルハンス島に残されたインスリンです。 </li><li>インスリンレベルをアッセイするために、従来のELISAを使用して、先に説明した3。 </li></ol>

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The authors declare no competing financial interests for the described work.

ここでは、従来の灌流のためには小さすぎる膵臓から膵島単離にステップバイステップのプロトコルを提供します。このような等 β細胞の精製、遺伝子発現解析、膵島β細胞の成熟、増殖、細胞ストレス応答、細胞生存率、代謝、機能GSISの維持、などのすべての島ベースの研究のための準備ができて膵島をもたらすことが期待されます。これは、我々の知る限りに、新生仔マウスから?...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table><tbody><tr><td>Collagenase Type IV</td><td>Sigma Aldrich, St Louis, MO</td><td>C5138</td><td></td></tr><tr><td>1x HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech/Cellgro, Manassas, VA</td><td>MT21020CV</td><td>If HBSS wihout Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; is obtained, CaCl&lt;sub&gt;2&lt;/sub&gt; and MgSO&lt;sub&gt;4&lt;/sub&gt; can be added to 1.26 and 0.5 mM, respectively.&amp;nbsp;</td></tr><tr><td>RPMI 1640 w/o glucose</td><td>Thermo Fisher Scientific/Life Technologies, Waltham, MA</td><td>11879-020</td><td>Glucose needs to be added to specific levels to not interfere with subsequent islet usage.&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma Aldrich, St Louis, MO</td><td>G-7021&amp;nbsp;</td><td></td></tr><tr><td>polysucrose and sodium diatrizoate solution</td><td>Sigma Aldrich, St Louis, MO</td><td>Histopaque-10771</td><td></td></tr><tr><td>Stereoscope</td><td>Carl-Zeiss, Oberkochen, Germany</td><td>Stemi2000</td><td></td></tr><tr><td>Stereoscope</td><td>Leica, Wetzlar, Germany</td><td>Leica M165</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5417C</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>15-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-666</td><td></td></tr><tr><td>50-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-658</td><td></td></tr><tr><td>Precision balance</td><td>VWR, Radnor, PA</td><td>VWR-225AC</td><td></td></tr><tr><td>Microfuge tubes</td><td>VWR, Radnor, PA</td><td>87003-294</td><td></td></tr><tr><td>Pipetman P1000</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123602</td><td></td></tr><tr><td>Pipetman P20</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123600</td><td></td></tr><tr><td>100 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-088</td><td></td></tr><tr><td>60 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-168</td><td></td></tr><tr><td>12-well plates</td><td>VWR, Radnor, PA</td><td>665-180</td><td></td></tr><tr><td>Scissor</td><td>Fine Scientific Tools, Foster City, CA</td><td>14080-11</td><td></td></tr><tr><td>Tweezers</td><td>Fine Scientific Tools, Foster City, CA</td><td>5708-5</td><td></td></tr><tr><td>CD1 mice</td><td>Charles River Laboratories, Wilminton, MA&amp;nbsp;</td><td>CD-1</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>C57BL/6J</td><td></td></tr><tr><td>B6CBAF1/J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>B6CBAF1/J</td><td></td></tr></tbody></table>

effective isolation of functional islets from neonatal mouse pancreas

大型哺乳動物の膵臓からの灌流ベースの膵島分離プロトコルは十分に確立されています。このようなプロトコルは容易に準備ができて、コラゲナーゼ注入とその後の組織潅流を可能にする膵管の大きなサイズのため、多くの研究室で行われています。灌流は小さな膵臓で容易に達成可能ではないので、コントラスト、小さな膵臓から膵島単離では、新生児マウスのような、挑戦的です。ここでは、視覚的な支援を受けて、新たに生まれたマウスからの膵島の相当数を回復する詳細な簡単な手順について説明します。新たに切開全膵臓を微量遠心管中、37℃で、ハンクス平衡塩溶液（HBSS）中に溶解し0.5ミリグラム/ mLのコラゲナーゼIVで消化しました。チューブは、組織の分散を助けるために、定期的にタップされました。組織の大部分を1mmの周りの小さいクラスタに分散させたときに、溶解物を、10％ウシ胎児血清（FBS）で培養培地で3〜4回洗浄しました。アイレットクラスター、認識腺房組織を欠く、その後ステレオスコープを解剖下で回収することができます。新たに生まれたマウスの膵臓あたりの大型小島に80小 - この方法は、20を回収します。これらの島は、インスリン分泌、遺伝子発現、および文化を含むほとんどの考えられる下流のアッセイに適しています。インスリン分泌アッセイの例は、単離プロセスを検証するために提示されています。遺伝的背景と消化の程度が収量を決定する最大の要因です。それは内分泌外分泌分離に役立つように、高い活性を有する新たに作られたコラゲナーゼ溶液が好ましいです。洗浄中のすべてのソリューションやウシ胎児血清中の陽イオンの存在[カルシウム（Ca 2+）とマグネシウム（Mg 2+）] /ピッキングメディアは、適切な整合性を持つ島の良好な収率のために必要です。良好なコントラストおよび倍率の解剖スコープにも役立ちます。

各小マウスの膵臓から80小島 - 最適な条件の下では、提示された方法は、20を得ることができます。この数は、マウスの遺伝的背景、年齢に依存し、島の大きさは、回収されます。一般的に使用され、CD1の中で繁殖させたアウトとC57BL / 6J純血マウスCD1とC57BL / 6の間のハイブリッドまたは商業B6CBAF1 / Jマウスが何よりも小さいサイズの小さい島を生産します。一般的にピ...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

我々は、新生児マウスから無傷の膵島を単離するために、本明細書のプロトコルを記述します。膵臓は、部分的に洗浄し、ハンドピッキング続いて、コラゲナーゼで消化しました。 20から80膵島クラスタは、いくつかの島の研究に適しており、新たに生まれたマウスから膵臓ごとに取得することができます。

新生児マウスの膵臓からの機能性膵島の効果的な分離

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Research

JoVE Journal

Developmental Biology

10.0K ビュー.  Vanderbilt University Medical School. 我々は、新生児マウスから無傷の膵島を単離するために、本明細書のプロトコルを記述します。膵臓は、部分的に洗浄し、ハンドピッキング続いて、コラゲナーゼで消化しました。 20から80膵島クラスタは、いくつかの島の研究に適しており、新たに生まれたマウスから膵臓ごとに取得することができます。 

ビデオ: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

マウス膵島の単離と被膜下腎移植のための方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

医学

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

部分的に膵臓を摘出された患者からのヒト膵島の単離

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

糖尿病マウスの精巣上体脂肪細胞における膵島支持骨格移植

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工学

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.

LEPR<sup&gt; DB</sup&gt; 2型糖尿病のマウスモデル：無傷の膵島の膵島単離およびライブセル2光子イメージング

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, ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

免疫・感染学

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

ハイコンテンツ<em&gt;インビトロ</em&gt;膵島β細胞の複製ディスカバリー・プラットフォーム

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

コラゲナーゼと中性プロテアーゼの精製の組合せを用いて単離ランゲルハンスマウスの膵島

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

生物学

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.

マウス モデルにおける膵島の門脈内移植

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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 ...

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.

新生児マウスの膵臓からの機能性膵島の効果的な分離

要約

さらに動画を探す

この動画の章

マウス膵島の単離と被膜下腎移植のための方法

部分的に膵臓を摘出された患者からのヒト膵島の単離

糖尿病マウスの精巣上体脂肪細胞における膵島支持骨格移植

LEPR

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

ハイコンテンツ

コラゲナーゼと中性プロテアーゼの精製の組合せを用いて単離ランゲルハンスマウスの膵島

マウスモデルにおける膵島の門脈内移植

マウス膵島の単離と細胞内cAMP測定法

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

新生児マウスの膵臓からの機能性膵島の効果的な分離

要約

さらに動画を探す

この動画の章

マウス膵島の単離と被膜下腎移植のための方法

部分的に膵臓を摘出された患者からのヒト膵島の単離

糖尿病マウスの精巣上体脂肪細胞における膵島支持骨格移植

LEPR

マウスの膵島細胞ベースの糖尿病誘発環境化学物質のスクリーニング

ハイコンテンツ

コラゲナーゼと中性プロテアーゼの精製の組合せを用いて単離ランゲルハンスマウスの膵島

マウス モデルにおける膵島の門脈内移植

マウス膵島の単離と細胞内cAMP測定法

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

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