私たちは、動物のケアとの技術支援のためにAniCanのスタッフ（CRCL、リヨン）に感謝。この作品は、協会注ぎラルシェルシュシュールルがんによって、研究所国立デュがんによって、およびからの奨学金によって研究所ナショナル·デ·ラ·サンテエ·デ·ラ·ルシェルシュMédicale（INSERMアベニールプログラム）、リーグ国立結界伤害防护结界ルがん、によってサポートされていました研究所国立デュがん（JG）から、フランスのMinistereはドゥEnseignement高等音楽エトデ·ラ·ルシェルシュ（RMPとDFV）から協会注ぐラルシェルシュシュールル蟹座（からリーグ国立結界伤害防护结界ルがん（JG）、 DFV）。

すべての手順は、政府機関（ &quot;ComitéD&#39;評価·コミュニケーションズのauセンターレオン·ベラール、ドゥENS、auのPBESらのauラボラトリーP4àL&#39;Animalerieデ·トランジット&quot;（CECCAPP））の規制の下で倫理委員会によって承認された。マウスは &quot;Plateforme AniCan、センターレオン·ベラール&quot;（リヨン、フランス）で、特定の病原体を含まない動物施設で維持や制度のガイドラインに準拠して処理されていました。 手順の概略を図1に示されている。 1。膵臓の解剖と引き裂くこと（0日目） 非常に迅速な解剖は、抽出の最適な収量のために重要であると培養中の細胞の良好な生存能力を保証する。膵臓の分離に必要な時間を低減するために、すべての器具および装置は、マウスの安楽死前の準備でなければならない。 <ol><li><html> CO 2窒息または頸椎脱臼によりマウスを安楽死させる。 </li></ol>このステップでは、すべての手順は、無菌解剖機器と滅菌雰囲気（微生物安全キャビネット、レベルII）の下で行われなければならない。 <ol start="2"><li>マウスを修正し、70％エタノールでマウスの腹部をスプレー。任意の解剖ハサミとピンセットで、陰部にV字型の切開を行い、完全に腹腔を開くためにダイアフラムにそれを続けています。 </li><li>ダイアフラムに対して肝臓ローブを置き、体腔が十分開いている場合、彼らはそこに残っているはずです。左手に腹腔外腸とコロンを引き、直腸を見つける。湾曲したピンセットや解剖ハサミ、グラブとセクションと直腸。 </li><li>鉗子の同じペアを使用すると、慎重に左手に腸を引っ張って完全に直腸から胃に腸をアンロール。 </li></ol>このステップでは、膵臓は、胃や腸の初めの間に小さなストリップとしてENT &quot;&gt;を区別することができます。脾臓とのライゲーションはそのまま残ります。 <ol start="5"><li> Noyes氏はさみとピンセットを使用して、慎重に腸に沿って膵臓をカットし、消化管の他の部分から脾臓でそれを解放する。 </li><li>脾臓とセクションそれ（ 図2）に接続されている膵臓をつかむ。 </li></ol>このステップでは、まだ腸間膜脂肪組織および/または他の隣接組織（脾臓、腸など）セルラ汚染を避けるために、膵臓で収集されなかったことを確認してください。 残りの手順は、すべてのバッファは単一腺房細胞における膵外分泌組織の完全な解離を回避するために、カルシウムイオンCa 2 +のキレート剤なしで準備する必要があります。 <ol start="7"><li>ハンクス平衡塩類溶液（HBSS）1Xに二度膵臓をすすぐ。 </li></ol>このステップでは、などの脂肪組織では、沈んでしまう膵臓に反して、それはまだ膵臓に付着汚染白色脂肪組織を可視化し、迅速に除去することが容易に可能であるフロートします。 膵臓細胞培養施設に輸送する必要がある場合は、1×HBSSで氷上に維持しなければならない。 <ol start="8"><li> HBSS 1X 5mlのを含む滅菌ペトリ皿で​​膵臓を転送します。 Noyes氏はさみやメスを使用して1〜3のMM 3（ 図3A）の小片で膵臓をスライス。 </li></ol> 2。膵臓の酵素および機械解離（0日目） <ol><li>滅菌した50mlポリプロピレンチューブにそれらを転送します。 </li><li> 450 XGと4で2分間遠心℃、吸引し、細胞の破片や血液細胞を除去するために、上清を捨てる。 </li><li>コラゲナーゼIA溶液10ml（HBSS 1×10 mMのHEPES、collagenaの200 U / mlを入れを追加するそれIA、および膵臓部にトリプシンインヒビターを0.25 mg / ml）で。 25ミリリットル血清ピペット使用し、25cm 2のフラスコに移す。 37℃で20〜30分間それをインキュベート℃にこの時間（5分ごと）に、減少サイズの滅菌ピペット（25、10、および5ミリリットル血清ピペット）で、精力的に10回程度往復膵臓断片を移動することによって、機械的解離を行う。 </li></ol>このステップで、それはしばしば膵臓切片の酵素解離の程度を監視することが不可欠である。 <ol start="4"><li>膵臓組織が ​​十分に解離すると思われる場合には（膵臓フラグメントの消失に、溶液の増加した濁度に応じて）（ 図3B）、冷を10ml添加して酵素反応を停止する（HBSS 1×5を含有する洗浄液を緩衝％ウシ胎児血清（FBS）および10mM HEPES）。 </li><li>滅菌した50mlポリプロピレンチューブとCENTRにそれを移す450×gで2分間、4℃のためifuge慎重に吸引し、コラゲナーゼIAソリューションを削除するには、上清を捨てる。 </li><li>バッファリングされた洗浄溶液10mlでペレットを再懸濁し、洗浄する。 450 XGと4で3分間遠心℃、慎重に上清を吸引して捨てる。このステップを2回以上繰り返します。 </li></ol> 3。腺房（0日）を分散し、濾過および播種<ol><li> 2.5％のFBS、1％ペニシリン - ストレプトマイシン混合物（PS）、トリプシンインヒビターを0.25 mg / mlの、組換えヒト上皮成長因子の25 ngの/ mlの（EGF）を含有するWaymouthの媒体から7 mlの細胞ペレットを再懸濁する。 </li><li>ろ液は、非消化断片（管、血管、およびランゲルハンス島）を保持するフィルタが100μmを通過させることにより、細胞混合物。膵臓腺房構造は（10-15細胞の腺房）を通過します。 </li><li> PS、トリプシンINH、Waymouthの含有培地FBS 6mlのとフィルターをすすぐibitor、およびEGF。 </li></ol>このステップの後、細胞は、任意の腺房解離を避けるために、非常に注意深く取り扱われなければならない。 <ol start="4"><li>シード6ウェル培養皿で孤立房（ウェル当たり2ミリリットル）（ 図3C）。文化それらの中で37℃、5％下C（v / v）のCO 2雰囲気。 </li></ol>このステップの後、腺房細胞が懸濁液中で培養する。 4。腺房細胞培養（目1〜10） <ol><li>二十四時間一晩付着（ 図4）している汚染物質の細胞と細胞の残党を排除する、新しい6ウェル培養皿に房を（懸濁液中の）を転送、後。 </li></ol>細胞培養では、数日間または実験条件は、細胞が単層培養が必要な場合、それはマトリックス足場上の転送や種子房に推奨され拡張される必要がある場合。 <ol start="2"><li>見前日マトリックスのサポート（0日）に鼎、コートI型コラーゲン（5μgの/ cm 2）を、6ウェル培養皿。各ウェルにI型コラーゲン溶液（0.02 M酢酸中50μgの/ mlで、0.2μmのフィルター）を1 mlを加え、37℃で1時間の間に、プラスチック上の受動吸着°C（または一晩、それを可能にする4℃で）。 </li><li> I型コラーゲン溶液を吸引除去し、リン酸緩衝生理食塩水1Xで二回うまくコーティングをすすいでください。 </li><li>使用前（微生物安全キャビネットの下）は、少なくとも12時間を乾燥させるだけでなくコーティングできます。 </li><li>タイプ前述のように同じ条件で私はコラーゲンコート6ウェル培養皿や文化、それら（37℃5％下（v / v）のCO 2雰囲気）に隔離された主要な腺房を（ステップ4.1で得られた）を転送。細胞は2日間のI型コラーゲンの基板に付着する。 </li><li> 3日目に、付着していない非生存細胞を排除するために培地を変更してください。文化の中で時間とともに、細胞が徐々にSP意志コラーゲン含有担体上でお読みください。 3日ごとに（ 図5）培地を変更します。 </li></ol>得られた単離された腺房細胞は、チャンバを計数トーマセルを使用して、完全な機械的解離した後、計数することができる。孤立した腺房細胞はその後文化の中で維持することができないことに注意してください。 取得した腺房文化の質は、 トリプシノーゲン 、 膵臓転写因子1サブユニットアルファ 、またはカルボキシペプチダーゼA1（免疫または免疫蛍光実験によって）として腺特異的マーカーの発現を確認することにより制御することができます。

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

このプロトコルでは、膵腺房細胞を単離するための手順を説明する。この方法は、1時間未満で動物枚以上20×10 6個の腺房細胞を単離することができる。その迅速かつ簡単な実装（のような多くの10と膵臓は独立並行して実験ごとに処理することができます）のおかげで、このプロトコルは、既存の単離法3-5,9-12,17の間の良好な妥協点として表示されます。 <p class="jov...

膵外分泌組織に取り組んで研究室のために頻繁に遭遇する問題は、十分な長さの長期的な実験を可能にするために一定の期間、in vitroで腺房細胞を培養することの難しさである。 このような培養系の開発を阻害する要因の一つは、それらが膵臓細胞の単離の間に放出されるとき文字通り膵臓組織を消化し、解糖タンパク質分解、および脂肪分解酵素の高い含有量に起因する実験的な操作に膵臓組織の固有感度である。 第二の要因は、その分泌特性を失い、例えば膵管細胞または肝細胞様細胞1のような他の成熟細胞への分化転換する傾向腺房細胞のin vitroでの著しい可塑性である。 インビトロにおいて 、この細胞可塑性実験条件によって変化する（このような培養培地の組成物として）2 </&gt;（商標）と膵外分泌細胞の1のための適切な培養条件の設計に複雑さの度合いを紹介します。 いくつかの方法がモルモットの膵臓から第3-5、腺房細胞の単離および培養のために開発されてきた。当初、これらのプロトコルは、精力的な、機械的解離によって究極の分離と、コラゲナーゼ、キモトリプシン、およびプロテアーゼカクテルと共に膵臓組織の消化を関与。このように分離された膵臓細胞、特に異常な構造的および機能的特徴、構造及びそれらの頂端膜受容体に著しい損害の損失を示した。単離された細胞は、わずか1〜2日間に実行可能なままであった。 分散した腺房の調製は、細胞膜を維持表面受容体への損傷を制限するので、分泌6-8に応答して分泌外分泌を向上させる、それらの細胞内および細胞間のアーキテクチャを維持する。 RESUとしてLTは、この方法は、in vitroで 7-10日に腺房細胞の生存を延長の主な利点を提供しています。ギャップ結合による細胞結合を含む細胞間連絡先のメンテナンスが外分泌膵臓腺房細胞表現型13の重要な決定因子であるので、さらに、この方法は、現在9-12腺房細胞の分離に好適である。 腺房細胞と管細胞への分化の分化は、積極的な膵外分泌癌14の起源のために提案されたメカニズムの一つであるため、分散した腺房モデルはまた、膵臓の可塑性とその後の分子メカニズムを研究するための適切なシステムである。さらに、遺伝子改変動物15,16及び遺伝子転写技術の開発（2アデノウイルス又はレンチウイルス形質導入、ナノ粒子の使用など）の使用と組み合わせて、このインビトロプライマリ腺房細胞モデルにおける缶種々の遺伝的機能不全は、腺房細胞分化または分化の調節に影響を与え、膵炎、前癌病変、および細胞可塑性の変化の開始を担当する分子事象のより良い理解を提供する方法を決定する上で非常に有用である。 分散した腺房の分離は、我々は文化膵臓腺房細胞への我々の研究室で使うアプローチです。私たちは、ここで使用方法を説明し、議論する。これは、腺房細胞の解離せずに機械的破壊に結合された膵臓組織（細菌コラゲナーゼ付き）の酵素的解離を伴う。ほとんどのプロトコルは、培養腺房が関与しながら、どちらか懸濁液中または特別扱いプラスチック基板上に、我々は長期化、細胞培養が必要な場合はマトリックス足場に後でそれらを播種、（24時間）だけ簡潔に懸濁液中で、それらを育てる。 このプロトコルは、分散した膵臓交流の急速な分離を（1時間未満の場合）ができますiniファイル、文化の中で一週間以上の持続可能な。それは、マウスの膵臓あたり20以上×10 6腺房細胞の単離を可能にします。そのシンプルさは、それが可能な独立して並行してできるだけ多くの10のような膵臓を処理することができます。現在利用可能なすべての他の膵外分泌モデルは、多くの遺伝的表示膵臓腫瘍から誘導される細胞内および細胞間の腺房の構造、したがって、単離された初代細胞の腺房表現型を維持することによって、このモデルは、分化メカニズムの研究のための最適なシステムを構成する変化は、細胞形質転換につながる。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>0.2 &mu;m filter</td>
			<td>Dutscher</td>
			<td>146560</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 &mu;m filter</td>
			<td>Beckton Dickinson</td>
			<td>352360</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 mm Petri dish</td>
			<td>Beckton Dickinson</td>
			<td>353003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1000 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1122-1830</td>
			<td></td>
		 </tr>
		 <tr>
			<td>20 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-1810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>200 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-8810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357535</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357543</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml polypropylene tube</td>
			<td>Beckton Dickinson</td>
			<td>352070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>6-well plate</td>
			<td>Beckton Dickinson</td>
			<td>353046</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetic acid 100%</td>
			<td>VWR BDH Prolabo</td>
			<td>20104.298</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase IA</td>
			<td>Sigma-Aldrich</td>
			<td>C2676</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Curved forceps, Dumont #7</td>
			<td>World Precision Instruments</td>
			<td>14188</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Dissecting scissors, straight</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Epidermal Growth Factor, human</td>
			<td>Promokine</td>
			<td>C-60180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol absolute (AnalaR Normapur)</td>
			<td>VWR BDH Prolabo</td>
			<td>20821.310</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps, Dumont #5</td>
			<td>World Precision Instruments</td>
			<td>14098</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Hank's Balanced Salt Solution 1x </td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES 1 M (pH 6.98-7.30)</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Incubator O2/CO2</td>
			<td>Sanyo</td>
			<td>MCO-19M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Matrigel</td>
			<td>Beckton Dickinson</td>
			<td>356234</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microbiological Safety Cabinet, level II</td>
			<td>Faster</td>
			<td>SafeFast Elite 212 S</td>
		 </tr>
		 <tr>
			<td>Noyes scissors, sharp/sharp tips, German</td>
			<td>World Precision Instruments</td>
			<td>500228-G</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin mixture</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffer Saline 10x </td>
			<td>Gibco</td>
			<td>14200067</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Company</td>
			<td>Pipet-Aid XP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scalpel</td>
			<td>Paramount Surgimed Ltd.</td>
			<td>Disposable Scalpel Size 23</td>
		 </tr>
		 <tr>
			<td>T25 flask, 25 cm2</td>
			<td>Sigma-Aldrich</td>
			<td>Z707481</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin inhibitor, from Glycine Max</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Type I collagen</td>
			<td>Beckton Dickinson</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Waymouth's medium</td>
			<td>Gibco</td>
			<td>31220-023</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and culture of mouse primary pancreatic acinar cells

このプロトコルにより、1週間以上の文化でそれらを維持すること、マウス膵臓腺房の​​迅速な分離を（1時間未満）で許可されます。以上20×10 6個の腺房細胞は、単一のマウス膵臓から得ることができる。このプロトコルは、独立して並行してできるだけ多くの10のような膵臓を処理する可能性を提供しています。それは腺房構造を維持するため、このモデルが十分にそれらの腺房分化の部分的または全体的な損失をもたらす多くの遺伝的変化を表示膵臓腫瘍から樹立細胞株とは対照的に、 インビトロで外分泌膵臓の生理機能を研究するために適している。

図1は、一次腺房細胞の分離のための&quot;分散&quot;腺房方法を図式化。厳密にプロトコルの間に尊重されなければならない重要なステップは、議論の部分で説明されています。 その除去を容易にするために、膵臓は、添付の脾臓（ 図2）と共に腹部から収集しなければならない。両方の器官が離れてカットする必要があり、まだ膵臓に取り付ける...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Primary Pancreatic Acinar Cells at JoVE.com

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

本書では、マウスの膵臓からプライマリー膵臓腺房細胞を単離し、培養するための迅速かつ便利な手順を説明します。この方法では、新鮮な主要形質転換/ノーマル膵外分泌細胞の生理機能を研究するための貴重なアプローチを構成している。

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 ...

isolation-and-culture-of-mouse-primary-pancreatic-acinar-cells

Research

JoVE Journal

Biology

35.3K ビュー.  Centre de Recherche en Cancérologie de Lyon. 本書では、マウスの膵臓からプライマリー膵臓腺房細胞を単離し、培養するための迅速かつ便利な手順を説明します。この方法では、新鮮な主要形質転換/ノーマル膵外分泌細胞の生理機能を研究するための貴重なアプローチを構成している。 

ビデオ: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

マウスの主栄養膜細胞と絨毛浸潤アッセイの分離

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

生物学

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture 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, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

カルシウムイメージング、細胞傷害測定、およびアデノウイルス感染の目的のための膵臓腺房細胞の調製

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

膵臓腺房細胞の分泌能力を評価する

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

ミニチュアブタからの原発性大動脈内皮細胞の単離と培養

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...