我々は肺を提供するためのマイアミ大学のライフアライアンスオルガン回復庁に感謝します。我々はまた、我々はまた、ガブリエルGaidoshに感謝図2Bおよび3-D、Cに図3（a）に実験に使用しmCherryをウイルス博士ベンGerovacとリサ・ノヴァクに示す実験に使用したDNA調製のために博士リサキュンチに感謝しますイメージング施設から、マイアミ大学眼科学科。 この研究は、NIH、CF財団博士マティアスSalatheへのフライトアテンダント医学研究所からの助成金が主催されています。

1.ステージ1：HEK 293Tの文化<ol><li>高グルコースのダルベッコ改変イーグル培地（DMEM）、10％（v / v）のウシ胎児血清（FBS）および1％（v / v）のペニシリン/ストレプトマイシンを添加することにより（HEK媒体という）HEK細胞の培地を調製します。 </li><li>コー​​トコラーゲンのコラーゲンI.ミックス30μlの私の蒸留水2ml中で1 10cmの組織培養皿。皿にミックスを広げ、37℃で2時間、5％CO 2インキュベートします。 </li><li>ミックスを削除し、15分間クリーンベンチ内の食器乾燥させます。 </li><li> 10ミリリットル中に1×10 6細胞の密度HEKメディアでコーティングされたディッシュ上にプレートHEK細胞と、37℃、5％CO 2でインキュベートします。 </li><li> HEK細胞50〜60％コンフルエント（ 図1a、メッキ後2-3日）である場合、密集度の2視覚的評価が十分にあるステージに進みます。いいえ定量化は必要ありません。 注：HEK細胞（上記参照）HEK培地中で培養されます。彼らはコンフルエントに達し、トンちょっと分割（ 例えば 1/5）以降の使用のためにダウンして凍結することができます。 </li></ol> 2.ステージ2：HEK細胞でのレンチウイルス（LVの）の生産と集中注：プロトコルは、BSL-2 USAで+（拡張BSL-2）の条件で、制度や政府の規制に沿って実行する必要があります。 <ol><li>カルシウム沈殿法を用いてトランスフェクト（リポフェクタミンおよび他のトランスフェクション剤がカルシウム沈殿法ほど効率的ではなかったです）。次のように手順が変更されたものの商業トランスフェクションキットの使用は、再現性を確保することをお勧めします。室温にすべての試薬を持参。これは、0日目です。 </li><li> HEK293細胞のそれぞれ10cmディッシュの場合は、エンベロープDNA PMD2-VSVGの4.5μgの、7.5μgのDNA pMDLg / pRREを包装、3.75μgのDNAのpRSV-REV、レンチウイルス発現ベクター10μgのDNA、2 Mカルシウム溶液の86μLを混ぜます700μlの最終容量に15mLの遠心管中に、滅菌水（後者の二つの溶液を、市販のトランスフェクションキットで提供されています）。 </li><li> （待っている間、ステップ4を実行）、賢明なドロップボルテックスしながら（クリーンベンチ内で）（トランスフェクションキットで提供さHBS）2×HEPES緩衝生理食塩水の700μlを添加し、30分間室温でインキュベートします。 </li><li>初期のインキュベーション時間の間に、スライド上のミックスの堆積物5μlの。ゆっくりと上下に焦点を当て、10倍の対物レンズと顕微鏡下での液滴を確認してください。微細な沈殿物が、リン酸カルシウム沈殿法を確認し、可視であるべきである（ 図1B）。 </li><li>混合するために上下HEK細胞メディアやピペットの10ミリリットルを追加することにより、30分後、沈殿を停止します。 </li><li>ステップ2.1から2.5からHEK細胞の皿を取り、そして培地（0日目）を削除します。皿の縁に沿って慎重にゆっくりとステップ2.5からの沈殿物の溶液を加えます。 </li><li>次の日（1日目）には、削除して、沈殿物を含む培地を破棄し、wはそれを置き換えますHEK媒体のi番目の10ミリリットル。顕微鏡下で微細な沈殿を確認してください：それは、細胞（ 図1cとd）の間の空きスペースに皿に表示されるはずです。 </li><li> 2日目に、シリンジを用いて40％のポリエチレングリコール（PEG）溶液3.8 mlを含有する15mLの遠心管に0.45μmのシリンジフィルターを通してフィルター媒体を集めます。ウイルスコレクションのすべてが完了するまで反転5Xは、（PEGを形成するために、沈殿したが、もはや5日以上の場合）、少なくとも24時間、4℃で混合し、ストアします。 </li><li> 4日目3日目と4上の手順を繰り返し2.8は、HEK培地と交換したが、10％の漂白剤で除染し、皿を破棄することはありません。 </li><li>遠心分離機すべてのコレクションチューブ（通常は5日目に一緒にすべてのコレクション）4℃で20分間、1650×gでは、ウイルスをペレット化します。削除し、上清を捨て、そして収集し、任意のPEG上清の残りを除去するために、4℃で5分間、1650×gで再びスピン。 </li><li> Resus保留アムホテリシンB 8なし気管支上皮成長培地（BEGM）の200μl中各管のペレット。それらを一緒にプールし、200μlにアリコート。 -80℃で保存ウイルス。 HIV-1のGag（P24）抗原ELISAアッセイを実行するために追加の10μLを分注し。 </li><li>市販のELISAキットを用い、製造業者のプロトコルに従ってp24抗原アッセイを行います。アッセイは、pg / mlで中のウイルスのp24の推定を与えます。 </li></ol> 3.ステージ3：文化初代HBE細胞<ol><li> 10 mlのBEGM培地でプレートHBE細胞はIを10cm組織培養皿コーティングされたコラーゲンで1×10 6細胞の密度で（100μlのアムホテリシンBで継代0細胞は、可能な真菌の汚染を除去するために使用される場合）。 37℃で培養し、5％CO 2。 </li><li>翌日、培地を除去し、4日間の合計（継代0細胞のための100μlのアムホテリシンBを含む）10ミリリットルBEGMと交換してください。インキュベーターに戻します。 </li><li> 4日後にAYS、一日おきにアムホテリシンBを変更メディアなしのみBEGMを使用します。 </li><li>細胞が80〜90％のコンフルエント（ 図2a;〜7日めっき後）である場合には、形質導入を進める（形質導入前の準備のために3.5に注意を払います）。再び、密集度の視覚的評価で十分です。いいえ定量化は必要ありません。 </li><li>形質導入の前に、コートコラーゲンIV溶液で透過性支持体の挿入は、無菌蒸留水で1/10に希釈しました。各インサートに200μlを添加して、クリーンベンチ内で一晩乾燥してみましょう。 </li></ol> 4.ステージ4：HBE細胞の形質導入<ol><li> 、メディアを削除するリン酸緩衝生理食塩水（EDTA / PBS）中に1mMのエチレンジアミン四酢酸中の0.1％トリプシンを3mlを添加し、37℃、5％CO 2で5分間インキュベートします。すべてのセルが切り離されているかどうかを確認するために、顕微鏡（10倍対物レンズ）の下で確認してください。ない場合は、さらに5分間インキュベーターに戻します。 </li><li>全ての細胞が分離されている場合、3を追加大豆トリプシンインヒビターのミリリットル（SBTIを1mg / ml）を、混合し、15mlの遠心管に移します。室温で5分間、460×gで回転させて細胞をペレット。 </li><li> BEGMの3ミリリットルに上清および再懸濁ペレットを削除して、カウントを進めます。 </li><li> BEGM400μlの（ 表2参照）内の1つの12ミリメートルの透過性支持体インサートあたり2×10 5細胞の割合で再懸濁HBE細胞（今継代1またはP1）。形質導入される透過性支持体インサートの量に基づいて、これらの数値を推定。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 表面 </td><td> 表面積 </td><td> 細胞計数 </td><td> メディア </td><td> ボリューム（メディア） </td></tr><tr><td> 10cmディッシュ</td><td> cm 2の52.7 </td><td> 1×10 6 </td><td> BEGM </td><td> 10ミリリットル</td> &lt;/ TR&gt; <tr><td> 12ミリメートルフィルター</td><td> 1.13センチメートル2 </td><td> 2×10 5 </td><td> BEGM </td><td> 400μlの</td></tr><tr><td>た24mmフィルター</td><td> 4.52センチメートル2 </td><td> 8×10 5 </td><td> BEGM </td><td> 800μlの</td></tr></tbody></table> 表2：細胞数あたりのメディアの外挿。 <ol start="5"><li> 4の感染（MOI）因子の多重度を使用して、細胞懸濁液にウイルスの適切な量を追加します。 </li><li> 2 / mlの最終濃度に臭化ヘキサジメトリンを追加します。 </li><li>先端区画に挿入透過性支持体あたりのミックス（BEGM、HBE細胞、ウイルスおよび臭化ヘキサジメトリン）の400μLを分注し、および側底コンパートメントに単独BEGMの1ミリリットルを追加します。該当する場合は、必ずコントロールおよび試験ピューロマイシン選択としてウイルスなしHBE細胞をプレート37℃で一晩インキュベートし、5％CO 2。 </li><li>翌日、レムオベ頂端および側底メディアは、PBSで洗浄し、気液界面（ALI）メディア8との両方の区画を交換してください。 </li><li>細胞がコンフルエントである場合、（気液界面を確立するとして知られている）頂端流体を除去。彼らは完全な成熟に達するまで、ボトムコンパートメント日おきに培地（ALI）を変更し続けます。通常3〜4週間後。 注：レンチウイルスベクターは、ピューロマイシンのような選択遺伝子が含まれている場合、細胞は、選択濃度曲線に従ってピューロマイシンを添加することによって選択することができます。 HBE細胞は、を1μg/ mlの濃度で形質導入した細胞の一貫した選択するための理想的であることが決定されています。しかし、この濃度は、他の細胞または条件ごとに異なる​​可能性があり、カーブを殺すことによって決定されるべきです。ピューロマイシンの添加は、メディアが選択遺伝子の完全な発現を可能にするALIメディアまたはそれ以降（2-3日）に置き換えられました後、早ければ1日として開始することができます。その時間1インサートにピューロマイシンを追加してください形質導入されていません。細胞が完全に分化されるまで、ピューロマイシンを添加することができます。 </li></ol>

<ol>
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The authors have nothing to disclose.

ここで説明するプロトコルは、100％の形質導入効率に近い保証します。しかし、この目標を達成するために重要な工程中のステップがあります。例えば、トランスフェクションプロセスの間に、日形質導入（ 図1b及びd）の後、形質導入ミックス（ドロップ）または皿の中の析出物の存在は、両方の良いトランスフェクションのために関連しています。沈殿物または?...

複製欠損、偽レンチウイルス（LV）の開発は、 インビトロ 1 で導入遺伝子を提供するために、安全かつ効率的な方法で研究者に提供してきました。それらの長期発現のために、これらのLVはまた、 インビボ 2,3 における治療剤の送達のために使用することができます。トランスファーベクター、パッケージングのgag / POL、パッケージ回転 、および水疱性口内炎ウイルス糖タンパク質（VSV-G）プラスミドエンベロープ：LVの製造は、いくつかのプラスミドを有するヒト胚腎臓（HEK）細胞の同時トランスフェクションを必要とします。 rev遺伝子は、別々のパッケージングプラスミド上にコードされているため、第三世代のパッケージングシステムは、それによって複製コンピテントレンチウイルスを生成するための組換えの可能性を低減する、第二世代のシステムよりも安全と考えられています。第二世代のパッケージングシステムが5倍高い総伝達ユニットを得ているが、元のウイルスゲノムの分割が作成します第三世代のシステムは、最小限3,4形質導入のレベルを減少しました。 細胞株は、より容易かつ効率的に形質導入することができるが、これらは、 インビボの組織5,6のより代表的なものであるため、研究者らは、一次細胞へのフォーカスをシフトしています。しかし、初代細胞は、形質導入に不応性です。完全に分化した気道上皮細胞の形質導入が報告されているが、全体の効率は15％7を超えていません。 ここで説明した高力価のLVの製造を可能にするプロトコルです。ステップバイステップのアプローチは、一次HBE細胞中にほぼ100％の形質導入効率を達成するために概説されています。具体的には、プロトコルは、3を成功するために尽力し、最適な培養条件を説明します。簡単に言えば、HEK 293T細胞を、強化緑色蛍光のEF-1プロモーター駆動発現を有するレンチウイルス発現ベクターpCDHを用いてトランスフェクションされていますタンパク質（EGFP）またはmCherryを、赤色蛍光タンパク質、および第三世代のパッケージングシステム。培地中に放出されたLVは24〜72時間後に回収されています。ウイルス粒子は、p24抗原の酵素結合免疫吸着アッセイ（ELISA）キットを用いて力価を推定する前に、ポリエチレングリコール（PEG）、ウイルス濃度の便利で簡単な方法で濃縮されています。 、4の（トリプシン処理された後）、感染の多重度（MOI）因子を取り付けるヘキサジメトリンブロミドを添加し、16時間インキュベートしながら、その後、未分化の一次HBE細胞（増殖培地（気管支上皮成長培地またはBEGM）8で形質導入され一晩）。次いで、細胞を分化させます。ウイルス導入遺伝子は（説明を参照してください、適切なプロモーターを用いて）、長期発現されるので、式は多列気道上皮への分化を通して維持されますまたは分化後（誘導性プロモーターを使用して）誘導することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>use at 10%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td>dilute 1:75 in distilled water</td>
		</tr>
		<tr>
			<td>Calphos</td>
			<td>Clontech</td>
			<td>631312</td>
			<td>Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O</td>
		</tr>
		<tr>
			<td>Polythylene glycol 8000</td>
			<td>VWR scientific</td>
			<td>101108-210</td>
			<td>stock of 40%</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A9528</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell 12mm</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C7521</td>
			<td>dilute 1:10 in distilled water</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>stock of 2mg/ml</td>
		</tr>
		<tr>
			<td>Puromycin (10.000x)</td>
			<td>Thermo Scientific</td>
			<td>A11138-03</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Alliance HIV-1 p24 ELISA</td>
			<td>PerkinElmer</td>
			<td>NEK050001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Scientific</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDH-EF1-MCS-IRES-Puro</td>
			<td>System Biosciences</td>
			<td>CD532A-2</td>
			<td>lentiviral expression vector</td>
		</tr>
		<tr>
			<td>pMD2-VSVG</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pRSV-rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td>packaging DNA</td>
		</tr>
	</tbody>
</table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

初代ヒト気管支上皮（HBE）細胞のインビトロ培養で気液界面条件を用いて気道細胞の分化と機能の過程を研究するための有用なモデルを提供します。過去数年間で、導入遺伝子の送達のためのレンチウイルスベクターの使用は、一般的な慣行となりました。特定の非HIV擬似型付けされたレンチウイルスと完全に分化した気道上皮細胞の形質導入の報告がありますが、全体的な形質導入効率は、通常15％未満です。ここで紹介するプロトコルは、レンチウイルスを生成すると、一次ヒト気管支上皮細胞を形質導入するために信頼性の高い効率的な方法を提供します。細胞が付着しながら4の感染因子の多重度で、未分化の気管支上皮細胞、気管支上皮増殖培地で形質導入を使用すると、100％に近い効率を提供します。このプロトコルは、高力価のレンチウイルスベクターの、ステップ・バイ・ステップ、準備と集中を説明し、第電子伝達プロセス。これは、初代ヒト気管支上皮細胞の高効率形質導入を達成するために最適な培養条件を決定した実験について説明します。

図1は、トランスフェクションプロセス中に細胞およびソリューションを評価する別の重要なステップを示している。 図1aは、従来のウイルス産生のためのトランスフェクションHEK293T細胞の最適なコンフルエンシーを示します。細胞をシャーレ全体に均等に分配されることが重要である。 図1bは、明視野光学と10倍の対物レンズ?...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

正常に伝達する初代ヒト気管支上皮細胞に必要な最適なレンチウイルスの生産および細胞培養条件

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

optimal-lentivirus-production-cell-culture-conditions-necessary-to

Research

JoVE Journal

Biology

19.2K ビュー.  University of Miami, Miller School of Medicine. Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

ビデオ: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

冷凍細胞から出発培養：ヒトES細胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

生物学

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

レンチウイルスの生産

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

外植片から成長した初代ヒト気管支上皮細胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

初代ヒト卵管上皮細胞のex vivoでの文化

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

強化されたバイオセーフティのためのポリマー複合エコトロピックレンチウイルスによるヒト細胞のトランスダクション

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

初代培養ラット副腎皮質細胞とステロイド ホルモン産生機能の試金

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

危険評価のための上皮細胞と原発性免疫細胞からなる多細胞ヒト歯槽モデル

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

オルガノイド培養のためのヒト肺上皮前駆細胞の単離と濃縮

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...