著者は、この研究で使用される人間の標本のドナー、RCWIH バイオバンク マウント シナイ病院/UHN 産科婦人科を感謝したいです。灰汁ラボ、特に博士キャロラインのダンク シュート法開発と彼女の助けのためのメンバーに感謝したいと思います。この作品はバロウズようこそ基金 (グラント #1013759) によってサポートされます。

胎盤は、選択科目の帝王を受ける労働女性ではなく、健康的な用語から収集されます。収集、処理、およびひと試料の使い捨ては、マウント シナイ病院倫理委員会のガイドラインに従います。各患者からの書面による同意を得た本研究は、マウント シナイ病院研究倫理委員会で承認されています。
1. 準備
注: すべての手順は、ヒューム フードの下で行われなければならないし、すべての手術用機器は発煙のフードで配置する前にオートクレーブで滅菌する必要があります。(ボトル、50 mL の管、等) 他のすべての材料は、70% エタノールで消毒する必要があります。常に摩耗保護具は、バイオハザード廃棄物 (白衣、手袋、長い髪を後ろで結ぶ、等) を使用する場合すべての回します。
<ol><li>
酵素消化ソリューション (最終的な解決: 200 mL)
	<ol>
<li>HBSS の 180 mL のピペット/- 500 mL ビーカーに滅菌攪拌磁石を追加し、室温で感動のプレートを配置。</li>
		<li>重量を量り、ゆっくりと順番に次の HBSS/ソリューションに追加: FBS、20 mL、400 mg コラゲナーゼ 2 ([final] = 2 mg/mL)、20 mg 大豆豆トリプシンインヒビター ([final] = 0.1 mg/mL)、30 mg DNase 1 ([final] = 0.15 mg/mL)、および 200 mg BSA ([final] = 1 mg/mL)</li>
		<li>中速に攪拌を設定し、10 ~ 20 分 (攪拌しながらスズ箔またはパラフィン フィルムでカバー) 攪拌混合物を許可します。</li>
		<li>プラスチックの漏斗を使用して 250 mL ガラス瓶の中に混合液を注ぎ、ヒューム フードに置きます。</li>
		<li>50 mL チューブ (合計 10 管) と使用するまで-20 ° C でストアに分注 20 mL プラスチック トップろ過ユニット (0.22 μ m メンブラン フィルター、500 mL)、ソリューションを渡します。</li>
	</ol>
</li>
	<li>
RPMI 1640 メディア (2% 洗濯ソリューションと完全な成長媒体の 10%)
	<ol>
<li>RPMI 1640 とヒューム フードのガラス瓶の FBS を組み合わせます。</li>
		<li>FBS 溶液の 2%、490 mL RPMI 1640、10 mL FBS を組み合わせます。</li>
		<li>FBS 溶液の 10%、450 mL RPMI 1640 と 50 mL FBS を組み合わせます。</li>
		<li>RPMI メディアと FBS (50 mg/mL 在庫、0.05 mg/mL 作業濃度) を含む前の手順から 500 mL ガラス瓶の中に normocin の 500 μ L をピペットします。</li>
		<li>使用するまで 4 ° C で 500 mL の瓶で 0.22 μ m のメンブラン フィルターとストアを介してメディアを渡します。</li>
	</ol>
</li>
	<li>実験を開始する前に 30 分の 20 mL の約数を配置、37 ° C のビーズのお風呂で酵素分解酵素液を凍結、2% を置く政府短期証券と 37 ° C のビーズのお風呂で 10% FBS RPMI 1640 メディア ロッキング水浴を入れます、37 ° C に設定、2 g および 4 ° C に温度制御された遠心分離機のセット</li>
</ol>2. 長期胎盤膜の脱落膜組織のコレクション
<ol><li>HBSS のボトルを配置 + +、HBSS-/- とヒューム フードの下のラックに 5 つの 50 mL チューブ。HBSS の 25 mL をピペット + + 1 つの管で。</li>
	<li>
手袋をはめた手を使って、満期胎盤から取り出す手術室劇場から輸送に使用されるコンテナー。おむつパッド (を母体側) の胎盤を置き、はさみとピンセットを使用して胎児膜を拡散します。
	<ol>
<li>ヒューム フードの重複のおむつパッドを置きます。</li>
		<li>おむつを使用して、母体側は表向きになり、胎盤を反転します。</li>
		<li>膜破裂のポイントを検索し、見つけ、展開しドラフト表面に平らになった膜を許可するように切開します。 注: 膜は胎盤から返したが、一度脱落膜顔になります後ろに羊膜層に、絨毛膜層に休憩を。</li>
	</ol>
</li>
	<li>絨毛膜と 25 mL HBSS を含む 50 の mL の管の場所から脱落膜組織をこすりプラスチック電池スクレーパーを使用して慎重に + +。</li>
	<li>適度な圧力で膜をこすり。絨毛膜汚染で起因するかもしれないのであまりプレッシャーは適用されません。同様に、これらはいくつかの脱落膜細胞を含んでいる小さな血塊を収集します。 注意: 脱落膜のコレクションの後胎盤バイオハザード プラスチック箱にパッケージ化、(あなたの制度的規則) に従って適切に廃棄する前に-20 ° C のフリーザーで凍結します。血まみれのおむつパッドを密閉ビニール袋に包まれ、バイオハザード安全ボックス内に配置する必要があります。</li>
</ol>3. 洗浄、酵素消化脱落膜組織の
<ol><li>優しく手で 50 mL のチューブを揺すり、無菌検体 (尿) コンテナー上休憩 250 μ m (250 μ m、サイズ 60 メッシュ) 金属製ふるいを通過によって収集された組織を洗浄します。</li>
	<li>HBSS で 2 回洗浄を繰り返す +/+ と 2 回 HBSS-/- 新鮮な管内各洗浄のため。(最終洗浄 HBSS-/-カルシウムやマグネシウムの HBSS の存在の除去では、+ +、次の酵素の消化力プロセスを妨害だろうそれ以外の場合)。 注意してください。洗浄のステップの間に絨毛膜組織汚染が脱落膜組織がうっすらとピンク色と白、密で、糸は非晶質、絨毛膜中、明らかにします。したがって、鉗子で絨毛膜汚染を簡単に削除することができます。</li>
	<li>必要に応じて、脱落膜組織が厚い場合、滅菌 10 cm 径シャーレに転送して皿に 2 つの反対のメスと組織のミンチに進みます。</li>
	<li>
組織/mL の酵素消化溶液の 100 mg を含む滅菌 50 mL チューブに洗浄ティッシュを置き (準備を参照してください)。
	<ol>
<li>基準点として脱落膜組織のレベルの 50 mL チューブに 5-10 mL マークに達することを確認します。</li>
		<li>全期間胎児膜から収集された合計脱落膜を消化する酵素消化液 (1.1 の手順で準備) 約 20 mL を使用します。</li>
	</ol>
</li>
	<li>パラフィン フィルム チューブのキャップ シール (チューブをしっかりとシールし、キャップとチューブの上部の周りパラフィン フィルムをラップ)、文化の下でフードし、振動水浴 (145 rpm で 20 分間 37 ° C で脱落膜組織を孵化させなさい、2 g)。</li>
	<li>インキュベーション後、パラフィンの膜を削除して消化組織の 70% エタノールを含むチューブの表面を殺菌してヒューム フードの下でもたらします。手で簡単にチューブを振る。</li>
	<li>金属ふるい (250 μ m、サイズ 60 メッシュ) を通じて新しい無菌検体容器に細胞懸濁液を収集します。酵素反応を停止する RPMI + 10 %fbs 含む 0.1 %normocin の等しいボリューム (20 mL) で希釈します。遠心分離のステップ 4.1 に直接進みます。</li>
	<li>必要に応じて、残りの未消化の組織を 20 ml の新鮮な酵素消化ソリューションの新しい 50 mL のチューブに配置し、消化 (20 分、37 ° C の水浴を揺れ) を繰り返します。</li>
	<li>2 番目の消化が必要な場合、氷 (滅菌を維持するパラフィン フィルムでカバー) に細胞懸濁液の最初の管を配置します。3.5 3.6 の手順を繰り返し、二つの細胞懸濁液を結合します。</li>
</ol>4. 単一細胞懸濁液を生成します。
<ol><li>遠心分離機の細胞懸濁液 (420 g、4 ° C、11 分)。</li>
	<li>上澄みを除去し、RPMI + 2 %fbs 含む 0.1 %normocin (「洗浄バッファー」) の 40 mL の細胞を再懸濁します。 注: 細胞ペレットに緩いと赤血球の混入によるゼラチン状、注意して上清を吸引します。手動ピペットの上部の段階の除去が必要な可能性があります。</li>
	<li>11 分の 4 ° C で 420 g で遠心分離を繰り返します。</li>
	<li>慎重に (前述の注の手順 4.2)、上清を削除 5 mL の洗浄バッファーで細胞ペレットを再懸濁し、同じ管で 35 mL の赤血球溶解バッファーを追加します。 注: 大規模なまたは非常に血なまぐさいペレットになった場合、それに分割できます赤血球溶解ステップの 2 つのチューブ 10 mL の洗浄バッファーを追加して、2 つの管に均等に分割各管に赤血球換散バッファーの 35 mL を追加して。</li>
	<li>20 分赤の血液細胞を溶解するインキュベーションの前後で簡単に渦尿管の氷の上を孵化させなさい。</li>
	<li>4 ° C で 11 分 420 g で遠心します。</li>
	<li>慎重に、上澄みを除去し、洗浄バッファーの 40 mL にペレットを再懸濁します。</li>
	<li>セルをセルの塊を除去する 70 μ m ナイロン フィルターに通過します。</li>
	<li>420 g で 4 ° C で 11 分間遠心</li>
	<li>上澄みを除去し、10 mL の完全培地で細胞ペレットを再懸濁します (RPMI 10% + FBS 0.1 %normocin を含む)。</li>
	<li>
トリパン ブルー色素排除の検定手順次のようを使用してセルをカウントします。
	<ol>
<li>文化フードの下でそっとトリパン ブルー色素と結合する前に徹底的に混ぜるために 3 x 上下細胞懸濁液をピペットします。1.5 mL チューブを準備細胞懸濁液、1:2 (結合トリパン ブルー溶液 20 μ L) と脱落膜細胞懸濁液 20 μ L を希釈します。慎重に (このソリューションは滅菌されていない) 数回上下ピペットでトリパン ブルー携帯ソリューションをミックスします。</li>
		<li>場所の上にガラス基板を顕微鏡のステージ上の検定。 注: 検定はトリプル ライン割った 9 つの大きな正方形をみるためにグリッドを顕微鏡スライドです。それぞれの大きな広場は 1 mm2の面積を有し、チャンバー内の液体の深さは 0.1 mm。したがって、それぞれの大きな広場を埋めることができる液量は 1 mm * 1 mm * 0.1 mm = 0.1 mm3= 10 の-4 mL。</li>
		<li>ゆっくりと全体スライド (ストップ混合物いっぱいでなくなる前に) 細胞の混合物を分散させるために毛細管をできるように、検定の溝にトリパン青細胞混合物の 10 μ L を追加します。</li>
		<li>(10 倍) で顕微鏡下で細胞を表示し、トリパン ブルー (これらは実行可能なセル)、検定の 4 つの大きい外側の四角を除外するすべてのホワイト/グリーン セルをカウントします。線に接するセルをカウントするときは、そのタッチの右と上の行が左と下の線に触れるものではなくのみカウントします。暗い青いセルはカウントされません。青い色は、トリパン ブルー色素が、簡単に細胞膜細胞質に侵入すると、細胞は死んでいることを示します。</li>
		<li>細胞懸濁液 (X) の 1 mL 中の生菌数の数を計算するには。 <img alt="Equation" src="/files/ftp_upload/57443/57443eq1v2.jpg"> 2希釈倍率を = 10,000 = 変換係数 (1 mL = 1 cm3 = 10、000 * 0.1 mm3)</li>
	</ol>
</li>
	<li>望ましい最終的な濃度 RPMI + 10% に脱落膜細胞を希釈 FBS (成長中)。 注: 組織培養めっき用ピペットの 2 * 106細胞/ウェル プラスチック 6 ウェル プレート、10 cm プラスチック プレート 10 * 106セルまたはガラス基板に 75,000 セルに。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+infection-induced+preterm+labour.">Cytokines and infection-induced preterm labour.</a> Reprodution Fertility and Development. 2, 499-510 (1990).</li><li>Yellon, S. M., Mackler, A. M., Kirby, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+leukocyte+traffic+and+activation+in+parturition.">The role of leukocyte traffic and activation in parturition.</a> Journal of the Society for Gynecologic Investigation. 10, 323-338 (2003).</li><li>Gomez-Lopez, N., StLouis, D., Lehr, M. S., Sanchez-Rodriguez, E. N., Arenas-Hernandez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+in+term+and+preterm+labor.">Immune cells in term and preterm labor.</a> Cellular &amp; Molecular Immunology. 11, 571-581 (2014).</li><li>Xu, Y., Plazyo, O., Romero, R., Hassan, S. S., Gomez-Lopez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+leukocytes+from+the+human+maternal-fetal+interface.">Isolation of leukocytes from the human maternal-fetal interface.</a> Journal of Visualized Experiments. 99, (2015).</li><li>Trundley, T., Gardner, L., Northfield, J., Moffett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+isolation+of+cells+from+the+human+fetal-maternal+interface.">Methods for isolation of cells from the human fetal-maternal interface.</a> Methods in Molecular Medicine. 122, 109-122 (2006).</li><li>Jividen, K., Movassagh, M. J., Jazaeri, A., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+for+establishing+primary+human+endometrial+stromal+cells+from+hysterectomy+specimens.">Two methods for establishing primary human endometrial stromal cells from hysterectomy specimens.</a> Journal of Visualized experiments. 87, (2014).</li><li>Pelekanos, R. A., Sardesai, V. S., Futrega, K., Lott, W. B., Kuhn, M., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+mesenchymal+stem/stromal+cells+derived+from+human+placenta+tissue.">Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue.</a> Journal of Visualized experiments. 112, (2016).</li><li>De Clercq, K., Hennes, A., Vrien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+endometrial+epithelial+and+stromal+cells+for+in+vitro+decidualization.">Isolation of mouse endometrial epithelial and stromal cells for in vitro decidualization.</a> Journal of Visualized Experiments. 121, (2017).</li><li>Zhang, J., Shynlova, O., Sabras, S., Bang, A., Briollais, L., Lye, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+and+activation+status+of+maternal+and+peripheral+blood+leukocytes+during+pregnancy+and+labour,+both+term+and+preterm.">Immunophenotyping and activation status of maternal and peripheral blood leukocytes during pregnancy and labour, both term and preterm.</a> Journal of Cellular and Molecular Medicine. 10, 2386-2402 (2017).</li></ol>

著者がある何も開示するには

ここで説明されているプロトコルは、非常に身近で簡単な一次脱落膜細胞を分離するための効果的な方法で採集された全体ひと満期胎盤の胎児膜時間とコストを示しています。このプロトコルの成功は 2 つの重要な要因、(1) (2) プロトコルを通して脱落膜細胞の処理とケアと胎児膜の絨毛膜層から脱落膜を削りの効率に依存しています。絨毛膜組織汚染がどれもが誤って含まれて脱落膜をこ?...

子宮内膜、最も活発な大人女性組織の 1 つは、劇的改造卵巣ホルモン、エストロゲン (E2) とプロゲステロン (P4) によって刺激への応答の各月経周期を経る。脱落膜、妊娠子宮内膜として知られているは、E2 支配的な増殖期に続く P4 駆動型分化の結果として postovulatory のフェーズの終了によって形成される非常に重要な生殖組織です。脱落膜細胞が分泌ホルモンの要因成功した胚盤胞の着床、胎児移植する母体を維持するため子宮胎盤インタ フェースの開発を担当。
エストロージェンは、注入と脱落膜らせん動脈のそれに続く改造に必要です。子宮内膜間質細胞は、月経周期1の後半の黄体期における P4 とキャンプの制御の下でエストロージェンを受けます。このプロセスは、血管と血管リモデリングと規制を人身売買白血球におけるその役割を示唆している間質全体に広がる周り開始です。この携帯の変換は、円形の形態、核サイズの増加と粗面小胞体とゴルジ装置2の拡大が特徴です。Decidualized 間質細胞は胚盤胞移植を支援パラクライン因子を作り出すことができる、多数のホルモン (プロラクチンすなわち)、血管新生因子、インスリン成長因子結合蛋白質 1 の分泌によって特徴づけられる(IGFBP 1), プロスタグランジン (PG) E (細胞内 cAMP の刺激)、サイトカイン、細胞外マトリックス成分と胎盤注入と開発3,4,5,6 に不可欠な栄養素.
脱落膜細胞の人口が decidualized の間質細胞のみで構成されていないも大きく、妊娠特有の脱落膜白血球ポピュレーションが含まれています。エストロージェンには、ローカライズされた一過性の浮腫とナチュラル キラー (NK) 細胞、T 細胞、樹状細胞、大食細胞の流入が含まれます。最大白血球サブポピュレーションが子宮 NK 細胞は、サイトカインの源である脱落膜・血管新生因子エストロージェン プロセスの援助し、の増加可能性のある浸潤母体のすべての白血球の約 50-70% を構成します。妊娠7全体の数です。大食細胞、免疫細胞の二番目に大きい集団をされて、移植部位周辺、妊娠8の間に増加します。彼らはサイトカインの源であり、成長因子などコロニー刺激要因 (CSF-1)9、腫瘍壊死因子 (tnf α) α10とプロスタグランジン (PG) E11。
妊娠中、長期的な労働者の前に、両名はサイトカインやケモカイン母体末梢血白血球の活性化と労働を開始する子宮の組織への移行のための責任の主要なソースです。動物研究ことを示した多数のプロ炎症性サイトカイン マウス脱落膜の調整、労働中によう TNF は、IL-6、イリノイ-12 と IL-1 b12。ひと脱落膜における炎症性サイトカイン IL-1 b、IL-6 と IL-8 (主要な好中球走化性因子) は労働労働13ではなくに比べて高い発現を展示します。これら分泌されるサイトカインの活性化、白血球の流入の結果脱落膜組織14;脱落膜浸潤子宮 4 倍大きく、この 2 つが隣接して子宮組織の活性化のカスケードを示すの前で長期的な労働者中、見ると脱落膜マクロファージと両方人間の好中球浸潤およびラットの増加15. PGs16子宮筋の収縮を同期をアクティブ化することを作り出すこれら白血球の浸潤、マトリックスメタロプロテアーゼ (Mmp) 膜を開始する破裂17,18, と同様子宮アクティベーション プロセス (「サイトカイン ストーム」) を増幅する炎症性サイトカイン。
初期妊娠期では、労働力の活性化に参加することで母体・胎児の公差を維持注入プロセスで重要な役割を再生などの脱落膜細胞の多くの重要な機能のため異なることができます中に発生します。妊娠。脱落膜の成熟の障害から再発着床障害再発妊娠の損失の原因 (1) 不妊につながる例えば、(2) 子宮内の成長の制限 (IUGR) と不適切な開発と脱落膜・胎盤や子宮脱落膜ジャンクション; 妥協血管変形の機能不全による妊娠中毒症(3) 早産、早期脱落膜活性化起因することができます。
研究生体内で人間の倫理的、実用的な制限と相まって、これらの主要な疾患に照らして体外より良い理解を目的とした分析のため不可欠です主のひと脱落膜細胞株を確立して妊娠合併症の臨床管理を改善します。したがって、我々 の研究の目的細胞の高と生存期間胎盤の胎児膜から収集したひと脱落膜細胞の分離を可能にするプロトコルを開発することでした。現在このプロトコルを細胞の脱落膜の特定のサブタイプの分離の明確に時間と費用の有効な方法について説明します様々 な体外分析に使用します。豊かさの特性と用語と最初または 2 番目の妊娠との比較で脱落のサブ集団の表現型人間の妊娠の中で自分の役割を定義することが重要です。

<table>
	<tbody>
		<tr>
			<td>Hank&#8217;s balanced salt solution with calcium and magnesium</td>
			<td>Prepared in facility (LTRI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution without calcium and magnesium</td>
			<td>Prepared in facility (LTRI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diaper pads</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>AL Medical</td>
			<td>2018-12-20.</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic disposable cell scraper (25 cm)</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mm (size 60 mesh) metal sieve</td>
			<td>Sigma-Aldrich</td>
			<td>S1020-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel with plastic handle (#21)</td>
			<td>Fisher Scientific</td>
			<td>08-927-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic petri dish (diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile specimen container (urine cup, 4.5 oz)</td>
			<td>VWR</td>
			<td>25384-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filter (70 mm)</td>
			<td>VWR/Corning</td>
			<td>21008-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrocyte lysis buffer</td>
			<td>Qiagen</td>
			<td>79217</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Reichert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 culture media</td>
			<td>Invitrogen</td>
			<td>11835-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin (50mg/ mL)</td>
			<td>Invivogen</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic top filtration unit (0.22 mm membrane, 500 mL)</td>
			<td>Millipore</td>
			<td>SCGPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2, lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy bean trypsin inhibitor, powder</td>
			<td>Sigma-Aldrich</td>
			<td>T9003-250mg</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase powder</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA powder)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope - Leica DMI 6000B</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Flow cytometer - Image Stream MK2</td>
			<td>Amnis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDEA software</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated Vimentin antibody</td>
			<td>R&#38;D Systems</td>
			<td>IC2105A</td>
			<td></td>
		</tr>
		<tr>
			<td>APC H7-conjugated CD45 antibody</td>
			<td>BD</td>
			<td>641399</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated Cytokeratin antibody</td>
			<td>MACs Miltenyi Biotec</td>
			<td>130-080-101</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP -conjugated Cytokeratin 7 antibody</td>
			<td>Novus</td>
			<td>NBP2-47941PCP</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor450 Fixable Viability dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>65-0863-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin primary antibody</td>
			<td>Santa Cruz</td>
			<td>sc-7558</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 primary antibody</td>
			<td>Dako</td>
			<td>M0701</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin primary antibody</td>
			<td>Dako</td>
			<td>M0821</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 7 primary antibody</td>
			<td>Dako</td>
			<td>M7018</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Santa Cruz</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat IgG</td>
			<td>Santa Cruz</td>
			<td>sc-2028</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10036</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 secondary antibody</td>
			<td>Fisher Scientific</td>
			<td>A-11058</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human decidual cells from the fetal membranes of term placentae

脱落膜、妊娠子宮内膜として知られている、非常に重要な生殖組織であります。脱落膜細胞は、主に decidualized の間質細胞と免疫細胞の構成は成功した胚盤胞移植、胎盤発育に欠かせないし、の役割を果たすホルモンや炎症性因子の分泌を担当されて、用語と早産で労働の開始。脱落膜を構成する異なる細胞集団の微妙なバランスの摂動から多くの妊娠合併症が生じる。脱落膜細胞の特定の種類の割合に変化がこれらの重要なプロセスを中断、妊娠、妊娠中毒症、子宮内胎児発育制限胚着床障害などの重篤な合併症の発症リスクを高めると早産。ここで説明されているプロトコルは、コストと時間的胎盤の胎児膜から収集した主のひと脱落膜細胞の隔離のための効果的な方法を示しています。酵素消化と脱落膜組織の穏やかな機械的破壊を組み合わせて、絨毛膜の汚染の事実上なしで脱落膜細胞の高収率が得られました。重要なは、孤立した脱落膜細胞が特徴付けられた (間質細胞 (55-60%)、白血球 (35%)、上皮 (1%) または (0.01%) 栄養膜細胞) マルチカラー イメージング流れ cytometry の試金によって確認された高い生存率 (80%) を維持。このプロトコルは脱落膜の parietalis に固有、最初と 2 番目の妊娠の胎盤に適応することができます。一度分離、脱落膜細胞は多数の妊娠合併症で別の脱落膜細胞集団の役割を理解することを目指して実験的アプリケーションを使用できます。

効率性と隔離されたセルの実行可能性を検証するため、彼らは 2 つの方法により特徴づけられた: 流れの cytometry と免疫細胞化学 (ICC)。4 細胞集団を対象としました。decidualized 間質細胞は抗ビメンチン抗体によって検出された汎白血球マーカー CD45 は脱落膜免疫細胞を識別するために使用された、サイトケラチンは、上皮・内皮細胞の検出に使用された、サイトケラチ...

Watch this Scientific Journal Video about Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae at JoVE.com

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

このプロトコルは、アプリケーション (すなわち免疫細胞化学、フローサイトメトリー、等) の様々 な使用することができます長期胎盤の胎児膜から収集した主のひと脱落膜細胞の分離方法を示しますを目指して妊娠合併症の異なる細胞集団の役割を研究。

用語胎盤の胎児膜から主のひと脱落膜細胞の分離

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

isolation-primary-human-decidual-cells-from-fetal-membranes-term

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14.1K ビュー.  University of Toronto. このプロトコルは、アプリケーション (すなわち免疫細胞化学、フローサイトメトリー、等) の様々 な使用することができます長期胎盤の胎児膜から収集した主のひと脱落膜細胞の分離方法を示しますを目指して妊娠合併症の異なる細胞集団の役割を研究。

ビデオ: Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

Isolation of Human Islets from Partially Pancreatectomized Patients

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

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

部分的に膵臓を摘出された患者からのヒト膵島の単離

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

医学

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

ヒト胎児膵臓細胞クラスターの単離、培養、およびイメージング

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

子宮摘出標本から初代ヒト子宮内膜間質細胞の確立するための2つの方法

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

マウス癌モデルからの循環腫瘍細胞の単離と伝播

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...