この研究は、潰瘍性大腸炎（R01AR069653〜Zhang LJ）および国立衛生研究所（5T32AR062496-03〜CA）によって支持された。

すべての動物実験は、UCSD機関動物管理および使用委員会の承認を受けています。 1.新生児皮膚由来の初代マウスKC単離および培養<ol><li> C57B / 6野生型マウス系統の生後0-2日の新生児をハサミを用いた断頭により犠牲にする。手首と足首の関節のすぐ上の手足を切断し、小さな穴を残して尻尾を完全に切断します。 </li><li> 皮膚全体を剥がすには、まず鋭いはさみを尾の穴に挿入し、身体の背中の中央線に沿って首の開口部に皮膚を切断します。次に、1つの鉗子を使用して、露出した身体と他の鉗子で皮膚をつかみ、身体から皮膚全体を1つの連続した動きで脚の切り株に静かに引き剥がします。 <ol><li>損傷のない部分として皮膚を剥がし、皮膚を破片に分解しないように注意してください。これは、ディスパーゼ工程中に細胞が失われるためです。 </li></ol></li> &lt;10cmのペトリ皿中の15mLの滅菌PBSで剥離した皮膚をすすぎ、次にKC増殖培地中に4mg / mLのディスパーゼを含む氷冷ディスパーゼ消化緩衝液を充填した2mLのチューブに各仔の皮膚を入れる（KC基礎培地は0.06mMのCaCl 2 、dGSおよび抗生物質/抗真菌薬を有する）。 注：複数の皮膚分離株を組み合わせて、ディスパーゼ溶液12 mLを含む1つの15 mLチューブ（チューブあたり最大5つのスキン）でインキュベートすることができます。ローテーター上で4℃の冷蔵庫で一晩皮膚をインキュベートする。 <ol><li>皮膚が折り畳まれていないことを確認してください。折り畳まれた部分がディスパーゼ溶液に不十分に曝されることになります。 </li></ol></li><li>翌日（ディスパーゼで12〜18時間後）、ディスパーゼ溶液と共に皮膚をペトリ皿に移す。過剰のディスパーゼを洗い流すために、15mLの滅菌PBSを含む新しいペトリ皿に各組織片を移す。 </li><li> 2対の鉗子を使用して、PBSから皮膚をつかみ、皮膚を持ち上げ、表側を下に、真皮側を上にして新しいペトリ皿に移す。肌のひだを注意深く引き伸ばし、ペトリ皿に完全に引き伸ばす。 </li><li> 新しいペトリ皿に各皮膚のためのトリプシンベースの消化溶液500μLを置きます。鉗子を使用して、表皮を下にして、表皮から真皮をゆっくり持ち上げます。生物を危険な物質として真皮を処分する。分離した表皮を移し、基底層を下にしてトリプシン溶液の各滴に浮遊させる。 <ol><li>表皮がトリプシン溶液で折りたたまれている場合は、上皮からの基底KCの効率的な消化を確実にするために、鉗子を使用して折り畳みを除去します。 </li></ol></li><li>穏やかに攪拌しながら水平シェーカー上で20分間室温（蓋を覆った状態）でトリプシン溶液中のペトリ皿上の皮膚をインキュベートする。 </li><li> 表皮あたり2mLの補充KC増殖培地（1平方インチあたり約1平方インチ表皮）をペトリ皿に加える。鉗子を使用して表皮を把握し、表皮を前後に激しくこすって表皮シートから単一細胞を放出させる。 <ol><li>細胞が表皮から分離するにつれて、中程度の濁りがますます激しくなるのを見てください。ペトリ皿を傾けて収集し、細胞懸濁液を新しいチューブに移し、残りの表皮シートを皿上に残す。 </li></ol></li><li> 2mLのKC増殖培地を添加した後、表皮シートをこすって2回以上繰り返し、同じチューブ内の細胞懸濁液を合わせる。 </li><li>適切な血清学的ピペットを使用して細胞塊を壊すために、細胞溶液を上下に穏やかに数回ピペットし、100μmフィルターに通して新しい50mL遠心チューブに移す。 </li><li>ろ過した細胞を180 x gで5分間遠心分離する。 </li><li>上清を吸引し、細胞ペレットを1mLの冷たいKC増殖培地/表皮に静かに再懸濁する。 </li><li>血球計数器で細胞を数える。 <br/&gt;注意：KCは懸濁液中で自発的に分化することができるので、細胞をできるだけ早く播種して細胞の最適な性能を確保する必要があります。細胞を氷上に置くことを推奨します。これは、KC自発分化の速度を低下させるためです。 </li><li> 細胞接着を促進する細胞外マトリックス（ECM）産物で予め被覆した培養皿中のKC増殖培地で5×10 4 / cm 2の密度で細胞を播種する。 注：我々は、市販のものを使用しています（例：ゼラチンベースのコーティング材料である付着因子（詳細については表を参照）。5％CO 2の加湿インキュベーターで37℃で細胞を培養し、一日おきに補充する。 <ol><li>コーティングのために、細胞播種前に10分〜2時間室温に加温された細胞付着コーティング材料で培養皿をコートする。直前のアタッチメント係数を削除する細胞懸濁液を添加する。 </li></ol></li><li>初期メッキの24時間後に培地を交換し、付着していない細胞を除去し、実験前に細胞が所望の集密度に達するまで毎日培地を交換する。 </li></ol> 2.成体テールスキンからの一次マウスKC単離<ol><li>動物施設の規定に従って、成体C57BL / 6マウス（好ましくは、6〜15週齢、男性または女性）を屠殺する。テールベースから身体の尾を切り取り、先端に目に見える穴ができるように尾の先端を〜2 mmカットします。 </li><li>尾の皮を剥がすには、まず鋭い刃を使って裾の皮を基部から尾の先端まで切断します。次に、1つの鉗子を使用して、露出した尾骨と他の鉗子で皮膚をつかみ、穏やかに1つの連続した動きで骨から尾の皮膚を剥がします。ミッドラインから剥がした肌を切り取って、各部分の長さが2〜3cm未満になるようにします。 </li><li> 剥がした肌をすすいでください10cmのペトリ皿に15mLの滅菌PBSを入れ、次に氷冷ディスパーゼ消化緩衝液（KC増殖培地中の4mg / mLディスパーゼ）で満たした2mLチューブに各尾から皮膚を置く。 注：複数の皮膚片を組み合わせて、ディスパーゼ溶液12 mLを含む1つの15 mLチューブ（チューブあたり5テールまで）でインキュベートすることもできます。 <ol><li>ローテーター上で4℃の冷蔵庫で一晩皮膚をインキュベートする。 </li></ol></li><li>単一の細胞懸濁液を尾の表皮から単離するために、ステップ1.4からステップ1.13を実行する。 </li><li>コラーゲンでプレコートした培養皿中のKC増殖培地中で10×10 4 / cm 2 （血球計算盤で計数）の密度で成体細胞を播種する。 注：市販のコラーゲンベースのコーティングキットを使用しています（詳細については、 表を参照）。細胞を37℃、5％CO 2の加湿インキュベーターで培養し、1日おきに新鮮な培地を補充した。一般に、c最終的な皿は、細胞播種の30分から1時間前にコラーゲンでコーティングされるべきである。本発明者らは、ゼラチンベースのコーティング材料である付着因子と比較して、コラーゲンでコーティングした場合、皿に成人のKC付着が有意に増強することを観察する。 </li><li>初期メッキの24時間後に培地を交換し、付着していない細胞を除去し、実験前に細胞が所望の集密度に達するまで毎日培地を交換する。 </li></ol> 3.高カルシウムによるKCのin vitro分化<ol><li>最終分化を誘導するために、培地中に0.2mMまでCaCl 2を添加する。 </li><li>あるいは、高カルシウムを添加する前に、低カルシウムKC基礎培地に増殖補助剤を添加せずに、一晩培養したKCを飢餓状態にする。 注：成長因子飢餓は早期分化への細胞のコミットメントを高め、早期分化マーカー、例えばK10 5 </。 </li></ol> 4.KBからのUVB照射細胞致死およびTNFα放出<ol><li> KC増殖培地（37℃、5％CO 2の加湿インキュベーター中）でマウスKCを培養し、細胞がコンフルエンシーに達するまで毎日培地を交換する。 UVB照射の直前に、増殖培地を除去し、500μLのPBSと交換する。次に、細胞を25mJ / cm 2 UVBまたはモックコントロール（UVBなし）に付す。 注：UVB照射は、以前10に記載されているように2個の8 W電球（312 nm）を有するハンドヘルドUVBランプを用いて行われます。 UVB照射後、PBSを吸引し、新鮮な培地を加える。 </li><li>測定及び6,8でCCK-8細胞生存率アッセイによって細胞生存率を定量化し、製造業者の説明書11以下の24時間後UVB治療。 </li><li>所望の時点でUVBで処理したKCから馴化培地を収集し、放出されたTNFαを測定する製造業者の指示7、12以下マウスTNF（モノ/モノ）ELISAキットによって馴化培地です。 </li></ol>

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著者は何も開示することはない。

皮膚の表皮は、身体を外部環境から分離し保護し、水分喪失、病原体、熱およびUV照射からの損傷に対する重大な障壁として機能する。 KCは表皮の優勢な細胞系統であり、表皮KCの初代培養は、インビトロでの障壁形成の生物学的プロセスおよび環境傷害に対するKCの応答を研究および理解するのに有用なツールである。 ここでは、初代表皮KCを新生児および成体マ...

皮膚は体の最も大きな臓器であり、最も外側の層は表皮である。表皮は、身体を環境から分離する無傷の表皮障壁を形成する上で決定的に重要な役割を果たし、したがって水分喪失を防ぎ、アレルゲン、病原体およびUVB曝露などの環境傷害から防御する。表皮は、未分化基底ケラチノサイト（KC）の単一層から基底層、棘突起層、顆粒層および角質層からなる多層重層化上皮に発達する。表皮幹細胞および中継増幅細胞の両方からなる基底KCは、増殖性および未分化性である。基本的なKCが細胞周期を終了すると、細胞は分化に拘束され、細胞 - 細胞接合の成熟および表皮透過障壁（EPB）の形成を伴って、表皮の表面に向かって徐々に移動する。棘状層のKCは、早期の差異を示すケラチン10（K10）のようなイオンマーカー; KCが顆粒層に移動すると、細胞はフィラグリン（FLG）、ロリクリン（LOR）およびインボルクリン（INV）などの後期分化マーカーを発現する。角質層では、KCは最終的に分化した角質細胞になり、最終的に新しい細胞がそれらに取って代わると落屑から流出する。 カルシウムは、表皮において最も生理学的に作用する物質であると考えられ、 インビトロおよびインビボで同様の方法で分化を誘発する。正常な皮膚表皮において、カルシウムイオンは、皮膚表面1、2、3に向かって濃度が増加し、特徴的な「濃度勾配」を形成します。カルシウム濃度は、最も低い副層（基底および棘の層）の低レベルから上部の顆粒層のピークまで上昇し、次いで最も表面の層（角質層）において無視できるレベルまで低下する。<html>カルシウム勾配は、カルシウム透過性がKC分化の重要な役割を果たすことを裏付ける成分透過性障壁の出現と同時に発生する。 インビトロでは 、低カルシウム（0.02-0.1mM）は単層としての基底KCの増殖を維持するが、高カルシウム（&gt; 0.1mM）はタイトジャンクション形成および誘導によって示されるように細胞の最終分化への迅速かつ不可逆的なコミットメントを誘導する基底KCS 4、5〜高カルシウム処理時LOR及びINVの。 障壁形成に加えて、表皮KCも皮膚の先天性免疫系の重要な構成要素である。 UVB照射または傷害時に放出される病原体または損傷関連分子パターン（DAMP）に応答して、KCはTNFα、IL6およびIFNβのような大量の炎症性サイトカインを産生し、免疫系の活性化をもたらす&gt; 6、7、8、9。 KCSから適切な炎症性シグナル伝達は、病原体のクリアランスに必要であるが、制御されない炎症性応答は、乾癬および酒6,8として自己炎症性皮膚疾患の発生をトリガすることができます。 全体として、KCは、無傷の皮膚バリアを維持し、病原体侵襲または環境傷害に対する免疫応答を開始する上で重要な役割を果たす。したがって、表皮KCの初代培養は、上皮生物学、KC分化、およびKC刺激による自然免疫応答を研究するための有用な技術である。主要マウス表皮KCの単離および培養は、KCの感受性および様々な外部刺激剤に対する感受性のために、困難なプロセスであり得る。ここでは、新生仔マウスの皮膚からKCを単離して培養する方法を説明します。成体マウスの尾の皮膚。成人のKC単離では、以下の理由により、この組織から十分な量の生存可能なKCを単離することが困難なため、マウス背側皮膚を使用しない：第1に、毛周期（休止期）の休止期における成人背側皮膚は、真皮からの表皮の非効率的な分離をもたらし、これは成功したKC単離のための重要なステップである。第2に、成人の背部皮膚上に存在する高い毛嚢密度は、真皮から表皮を分離することの困難さにさらに寄与する。その代わりに、我々は成体マウスKCsの供給源として尾皮膚を日常的に使用する。なぜなら、この上皮は表皮のKCの3〜5層でより厚いからである。それはまた、表皮の分離を妨げない、より低い毛嚢密度を有し、したがって、マウスの年齢および毛髪サイクリング段階に関係なく、成体マウス尾皮膚からのKC単離を可能にする。孤立した新生児タルKCをゼラチンコート培養皿に播種し、新生児対応細胞と比較して成体細胞が接着する能力が損なわれているため、コラーゲン被覆皿を用いて単離された成体KCを播種する。マウスKCを培養するために、低カルシウム基礎培地に、表皮成長因子（EGF）、ウシトランスフェリン、インスリン様増殖因子c1（IGF1）、プロスタグランジンE2（PGE2）、ウシ血清アルブミン（BSA）およびヒドロコルチゾンを含むdGSを補充する。最初の播種後2〜4日の間に、分化したKCの大部分は毎日の培地交換の間に洗い流すことができ、残りの接着細胞は典型的な敷石形態4を示し 、増殖し、早期分化マーカーK10を発現しない。

<table width="633">
	<tbody>
		<tr height="100">
			<td height="100" style="width:171px;height:100px;">C57BL/6 neonates or adult wildtype mice</td>
			<td style="width:177px;">Jackson Laboratory</td>
			<td style="width:119px;">000664</td>
			<td style="width:167px;">Wildtype mice (originally purchased from Jackson Laboratory) are breeded and maintained in animal vivarium at UCSD.</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">KC basal medium (EpiLife)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">MEPICF500</td>
			<td style="width:167px;">basal medium for keratinocyte culture with 0.06 mM CaCl2</td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">Defined Growth Supplement (dGS)</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S0125</td>
			<td style="width:167px;">defined growth supplements for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Dispase powder</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">17105041</td>
			<td style="width:167px;">enzyme to dissociate the epidermis from dermis</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Attachment Factor</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">S006100</td>
			<td style="width:167px;">gelatin-based coating material</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Coating Matrix</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">R011K</td>
			<td style="width:167px;">Collagen-based coating material</td>
		</tr>
		<tr height="80">
			<td height="80" style="width:171px;height:80px;">TrypLE</td>
			<td style="width:177px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:119px;">12604-013</td>
			<td style="width:167px;">A gentle trypsin-like enzyme to dissociate keratinocytes from epidermal sheet</td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">100 &#956;m Cell Strainer Nylon mesh</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">352360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="width:171px;height:60px;">CCK-8 cell viability Kit</td>
			<td style="width:177px;">Dojindo Molecular Technologies, Rockville, MD</td>
			<td style="width:119px;">CK04-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Mouse TNF (Mono/Mono) ELISA Set II</td>
			<td style="width:177px;">BD Biosciences, San Jose, CA</td>
			<td style="width:119px;">555268</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Corded Hand-Held UV Lamps</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">EB-280C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">8-watt UV tubes</td>
			<td style="width:177px;">Spectronics, Westbury, NY</td>
			<td style="width:119px;">BLE-8T312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:171px;height:40px;">Light Inverted Microscope for cell culture</td>
			<td style="width:177px;">ZEISS, Jena, Germany</td>
			<td style="width:119px;">Axio Observer</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:171px;height:20px;">Fluorescent Microscope</td>
			<td style="width:177px;">Olympus</td>
			<td style="width:119px;">BX41</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

ケラチノサイト（KC）は、皮膚の最外層である表皮の優勢な細胞型である。上皮KCは、UVB照射または病原体などの環境傷害に対するインタクトな皮膚バリアを形成することによって、またそれらの傷害に対して炎症反応を開始することによって、皮膚防御を提供する上で重要な役割を果たす。ここでは、新生児マウス皮膚および成体マウス尾皮膚からKCを単離する方法を説明する。本発明者らはまた、キレキシブウシ胎児血清（cFBS）と比較して規定された成長補助剤（dGS）を用いて培養条件を記載する。機能的には、新生児および成人の両方のKCが、高カルシウム誘発性の分化、タイトジャンクションの形成および層別化に対して高い応答性を有することを示す。さらに、培養された成体KCは、UVB誘発細胞死に感受性であり、UVB照射の際に大量のTNFを放出し得る。一緒に、ここに記載された方法は、インビトロモデルの設定のための研究者にとって有用であろう新生児マウスおよび/または成体マウスの表皮生物学を研究する。

高カルシウムは、新生児および成人のKCの最終分化を誘導した。 0.06mM CaCl 2で平板培養し維持した初代マウス表皮KCは、単層として増殖し、個々の細胞は、明確な細胞間空間を有する多角形を有し、コンフルエントになると、石畳の外観を示した（ 図1Aおよび図2A ）。 CaCl 2を0.2mMまで上昇させる...

Watch this Scientific Journal Video about Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin at JoVE.com

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

上皮ケラチノサイトは、機能的な皮膚バリアを形成し、外部環境傷害に対する宿主防御の最前線に位置付けられる。ここでは、新生児および成体マウスの皮膚からの表皮ケラチノサイトの単離および初代培養、およびケラチノサイトからの最終分化およびUVB誘発性炎症反応の誘導のための方法を記載する。

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-culture-primary-mouse-keratinocytes-from-neonatal-adult

Research

JoVE Journal

Biology

30.5K ビュー.  UC San Diego. 上皮ケラチノサイトは、機能的な皮膚バリアを形成し、外部環境傷害に対する宿主防御の最前線に位置付けられる。ここでは、新生児および成体マウスの皮膚からの表皮ケラチノサイトの単離および初代培養、およびケラチノサイトからの最終分化およびUVB誘発性炎症反応の誘導のための方法を記載する。 

ビデオ: Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

成体マウスの心筋細胞の単離、培養および形質導入

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...