著者はベッキー マルケス、センタラ肥満センター、患者スクリーニングと同意のプロセスに彼女の援助のための医療コーディネーターを認識したいです。この研究は、Anca + Dobrian に R15HL114062 によって支えられました。

東バージニア医学校制度のレビュー ボード委員会承認研究および研究で使用されるひと脂肪組織サンプルのコレクション。通知の書面による同意は、患者から採取しました。
1. バッファー、メディア、および計測器の準備
<ol><li>Krebs Ringer Bicarbonate-Buffered ソリューション (KRBBS) を準備: 塩化ナトリウム 135 ミリメートル、5 mM 塩化カリウム、硫酸マグネシウム 1 mM、0.4 mM カリウム リン酸水素、5.5 mM グルコース、1 mM アデノシン、0.01% 抗生物質/抗真菌薬ミックス (50 μ g/mL のペニシリン、ストレプトマイシン、ゲンタマイシンの 30 μ G/ml、アムホテリシン 15 ng/mL の 50 μ g/mL) 10 mm HEPES (pH 7.4 を =)。このソリューションは、組織の採取や消化をするたびに新鮮な準備されます。</li>
	<li>新鮮なコラゲナーゼ溶液を調製: KRBSS; で 1 mg/mL コラゲナーゼのタイプ 1、3 mL/g の脂肪を作る。事前組織消化前に水のお風呂で 37 ° C でコラゲナーゼの解決を温めます。</li>
	<li>Cd34 陽性細胞分離バッファーを準備: 2% ウシ胎児血清 (FBS)、1 mM EDTA 滅菌リン酸緩衝生理食塩水 (PBS)。</li>
	<li>脂肪細胞形成メディアの準備: DMEM/F12、5 %fbs、ペニシリン/ストレプトマイシン、ヒトインスリン (5 μ G/ml または 144 mU/mL) の 1.25 μ L/mL と 2.8 mM ロシグリタゾン (1 μ M) の 0.36 μ L/mL の 50 μ g/mL。</li>
	<li>100 mL のイソプロパノールにオロの 0.3 g を溶解することにより 0.3% オイル赤い O (オロ) 原液 (重量/体積) を準備します。</li>
</ol>2. 脂肪間質血管の一部分離
注: 研究は、病的肥満 2 型糖尿病 (T2D) と非糖尿病患者対象、18-65 歳以上センタラ代謝で減量外科センター (センタラ医療グループ、ノーフォーク、VA) 肥満症治療手術の断面のコホートを含まれています。1 型糖尿病は、慢性的な免疫抑制療法、抗炎症薬、thiazolinendiones、アクティブなタバコの使用、慢性や急性の感染症、または歴史を必要とする条件を含む自己免疫疾患が除外基準に含まれています。悪性腫瘍の最後の 12 ヶ月以内で処理。ダグラス T2D は、空腹時血糖 126 mg/dl 以上、200 ミリグラム/dL の以上 2 h 耐糖能をテストすると、血糖値や抗糖尿病薬の使用と定義しました。
<ol><li>肥満手術を受ける被験者からひと大網 (OM) と皮下 (SC) 脂肪 (AT) を収集します。</li>
	<li>組織抽出後すぐに室温でペニシリン/ストレプトマイシンの 50 μ g/mL のハンクの緩衝塩液を含む別のバイアルに OM と SC AT をしてください。</li>
	<li>慎重にきれいにし組織抽出後、ラボに到着すると、サンプル 70% エタノール採取はさみとピンセットを使用して、組織の線維化と焼灼のセクションを削除します。</li>
	<li>脂肪組織を圧迫し、コラゲナーゼの消化力のためのそれを 5 g (または少ない) 因数を分割します。</li>
	<li>はさみの 2 つのペアを使用して室温でシンチレーション バイアルにコラゲナーゼ溶液の 5 mL で AT の 5 g を細かくみじん切り。</li>
	<li>コラゲナーゼの解決の追加 10 mL を 15 mL 合計でみじん切りにした組織に追加します。</li>
	<li>連続振動で水浴中の 37 ° C で、1 h のサンプルを消化します。</li>
	<li>鈍い端に 20 mL の注射器に先端を削減します。</li>
	<li>250 μ m メッシュから 9 cm × 9 cm の正方形を切り取って鈍い端の注射器を使用して 50 mL の円錐管が途中で押し込みます。</li>
	<li>鈍い端の 20 mL シリンジと未消化の組織から脂肪細胞と血管の間質細胞を分離する 50 mL の円錐管に 250 μ m ナイロン メッシュをフィルターにサンプルを注ぐ。 注: 余分な線維化未消化材料によりメッシュが詰まって取得、50 mL コニカル チューブあたり 5 g 以上でを使用しないでください。</li>
	<li>KRBBS 10 mL でシンチレーションのバイアルを洗浄し、残留細胞を収集するフィルターを介して注ぐ。</li>
	<li>室温で 5 分間フィルターが適用されたサンプルをインキュベートします。 注: この手順は、フロート チューブの上部といくつか間質血管細胞の管の底に解決するために脂肪細胞を可能にすることが重要です。</li>
	<li>20 mL シリンジと間質血管の一部のレイヤーを削除し、きれいな 50 mL の円錐管に転送分注針で 20 x 6 を利用します。</li>
	<li>KRBBS 10 mL を追加し、室温で 5 分間ベンチに座るにサンプルを許可します。</li>
	<li>2.12-2.14 の手順を 2 回繰り返します。 注: これらの手順が浮動の脂肪細胞と間質の血管細胞の汚染がないことを確認します。</li>
	<li>以下の手順で説明するよう、両方の拠点、さらなる処理のため氷の上から間質血管の分数を維持します。 注: セル実行可能性に影響を与える可能性があります、1 h 以上の氷の上のセルを残してはいけない。脂肪細胞は長期保存用液体窒素でフラッシュ凍結することができます。 または実験用新鮮です。</li>
</ol>3. 脂肪組織の血管内皮細胞の分離
<ol><li>4 ° C で 5 分間 500 x gで間質血管の分数 (SVF) をスピンします。</li>
	<li>遠心分離機からサンプルを外し、慎重に; 上澄みを離れて注ぐSVF セルを含むペレットを維持します。</li>
	<li>軽く 5 mL の PBS で細胞ペレットを再懸濁します。 注: AT デポの 5 g 以上が複数の因数 (ステップ 2.4 参照) に処理された場合細胞ペレットを一緒に分かち合います。</li>
	<li>4 ° C で 5 分間 500 x gでサンプルをスピンします。</li>
	<li>上澄みを除去、1 mL の PBS、細胞を再懸濁します、セルをカウントします。 注: ここでは、セルの自動カウンターは、セルカウントに使われました。アクリジン オレンジ/propidium ヨウ化 (AO/PI) ソリューションの 12 μ L で 12 μ L の細胞懸濁液を混合することによって得られたセル実行可能性 (材料の表を参照してください)。各デポの AT のグラムあたりのセル数の平均値は: (a) OM デポ: 1.69 × 105 ± 4.51 × 104;(b) SC デポ: 105 ± 6.45 x 104× 1.24。両方の拠点から細胞の viability が > 90%。</li>
	<li>4 ° C で 5 分間 500 x gでサンプルをペレットします。 注: CD34 免疫選択プロトコルは手順 3.7 – 3.15 適応されました (材料の表を参照してください)。</li>
	<li>CD34 分離バッファーを 100 μ l 添加でペレットを再懸濁します。 注: 総細胞数次の再懸濁液ボリュームを必要とする: (a) < 2 x 10 の7セル: 100 μ L; でペレットを再懸濁します(b) 108–5 × 108セル × 2: 1 mL にペレットを再懸濁します。3.9-3.16 の手順で使用する試薬の量だったため縮小 < 2 x 10 の7セル。</li>
	<li>CD34 カクテル 10 μ L を追加し、室温で 15 分間サンプルをインキュベートします。</li>
	<li>磁気ビーズの 5 μ L を追加し、室温で 10 分間のサンプルをインキュベートします。</li>
	<li>各サンプルに 2.5 mL の CD34 分離バッファーを追加し、室温で 5 分間、磁石にサンプルを配置します。</li>
	<li>5 のための磁石を反転分離バッファーを注ぐ s。</li>
	<li>磁石から、サンプルを削除、手順 3.10 と 3.11 の 4 倍を繰り返します。</li>
	<li>磁石からチューブを削除し、2 mL の CD34 分離バッファーを追加します。</li>
	<li>4 ° C で 5 分間 500 x gで細胞をペレットします。</li>
	<li>CD34 分離バッファーの 1 mL の細胞ペレットを再懸濁し、セルをカウントします。 注: ここでは、AT のグラムあたりのセル数の平均値は: (a) OM デポ: 4.75 × 104 ± 3.36 × 104;(b) SC デポ: 104 9.53 × 10 ±3x 1.06。CD31 プラスチック immunobead プロトコル手順 3.16-3.34 (材料の表を参照してください) 適応されました。</li>
	<li>500 x gで 5 分で細胞をペレットし、バッファー B 250 μ、250 μ L の洗浄バッファーでペレットを再懸濁します。</li>
	<li>ボルテックス チューブ CD31 ビーズを徹底的に再懸濁します。</li>
	<li>CD31 ビーズの 20 μ L を追加します。 注: [集計] セルのためカウント > 1 × 106、ボリューム製造元の入力に応じて規模を縮小しました。</li>
	<li>30 分間、室温のロッキング サンプルをインキュベートします。</li>
	<li>ストレーナーを滅菌 50 mL の円錐管に接続します。 注: こし器の大きな開口部上にある必要があります。</li>
	<li>細胞分離前にストレーナーを平衡に洗浄バッファーの 1 つの mL を追加します。[ステップ 3.16 ストレーナーに生成された混合物を適用します。</li>
	<li>20 mL の容量の円運動で 5 mL の洗浄バッファーとストレーナーを洗浄します。コネクタを滅菌 50 mL の円錐管に接続し、ルアーロックを閉じます。</li>
	<li>ストレーナーをコネクタに取り付けます。こし器の壁に沿って洗浄バッファーの 1 つの mL を追加します。こし器の壁に沿ってアクティブ バッファー D の 1 mL を追加します。サンプルをゆっくり旋回し、室温で 10 分間インキュベートします。</li>
	<li>洗浄バッファーの 1 つの mL を追加します。ビーズから 10 倍上下ピペッティングにより細胞を分離します。 メモ: は、空気の泡を生成しないでください。</li>
	<li>ルアーロック-を開き、50 mL の円錐管に流入する剥離細胞を許可します。ストレーナー 10 を洗うたびに洗浄バッファーの 1 ml x。</li>
	<li>コネクタとストレーナーを破棄し、遠心分離機の 300 × g 10 分間、4 ° C でのサンプル文化の完全な EGM 2 メディアの 2 mL の細胞ペレットを再懸濁します。 注: この時点でのグラムあたりのセル数の平均値は: (a) OM デポ: 5.92 × 103 4.27 × 10 ±3;(b) SC デポ: 4.12 103 ± 2.1 x 103x。両方の拠点からの細胞の生存率は 90 パーセント以上だった。</li>
</ol>4. 血管内皮細胞脂肪細胞形成の誘導
<ol><li>37 ° C、5% CO2インキュベーターで完全な EGM 2 メディアの 6 ウェル培養処理板の細胞を成長します。メディアを交換して、電池が 80-90% の合流点に達するまで 3-4 日おき。 注: 人口倍増は 2-3 日です。</li>
	<li>100 mm ペトリ皿上それらをメッキすることによって細胞を展開し、1:3 回セルを分割します。この段階でセルが 2 の一節にあります。</li>
	<li>自動細胞を用いた細胞カウンター (材料の表を参照してください) の数。</li>
	<li>デポおよびそれらは 2 日間の EGM 2 メディアを完了で文化ごとに重複する井戸の確保 6 ウェル プレートに種子約 100-200,000 セルです。</li>
	<li>任意の成長媒体を離れて吸い出しなさいし、2 ml の 5% を DMEM/f12 キーの交換 FBS ペニシリン/ストレプトマイシン コントロールのための 50 μ g/mL 細胞治療細胞の脂肪細胞形成メディア (手順 1.4 参照) 2 mL に置き換えると。</li>
	<li>37 ° c 5% の加湿で細胞をインキュベート CO2インキュベーター 13 日間のそれぞれのメディアごと 3 〜 4 日の交換します。 注: 細胞治療の 4 日後に脂質を蓄積する開始されます。</li>
	<li>オロとナイルレッド公開メソッド15-17によると染色を行います。</li>
	<li>RNA 抽出のためのセルを分離するには、6 よく誘導プレートからメディアを取り出し、1 mL の滅菌 PBS で洗浄します。</li>
	<li>Guanidium チオシアン酸-フェノール-クロロホルム溶液の 350 μ L を追加 (材料の表を参照) もそれぞれ、5-10 分間室温でインキュベートします。</li>
	<li>細胞スクレーパーを使用してプレートから細胞をこすりし、セルを 1.5 mL 遠心チューブに転送します。 注: サンプルは、RNA の抽出、さらに後日処理-80 ° C のフリーザーで格納できます。</li>
	<li>適応の RNA 抽出を用いたサンプルから mRNA を抽出プロトコル (材料の表を参照してください)。</li>
	<li>リアルタイムのポリメラーゼの連鎖反応 (RT-PCR) 次のプローブを用いた遺伝子発現を評価するを実行: アディポネクチン、UCP-1、および CIDEA (材料の表を参照してください)。 注: ここでは、RT-PCR 法手続を実施した次の標準的なプロトコルを使用して: 変性 (95 ° C; 15 s)、アニーリング、および 40 のサイクルの拡張 (60 ° C; 1 分)。CTの値を持つ遺伝子を表すサンプル > 35 が検出されないと考えられていた。</li>
</ol>

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E., Woodcock, M. <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+characterization+of+microvessel+endothelial+cells+from+human+mammary+adipose+tissue.">Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue.</a> In Vitro Cellular &amp; Development Biology. 29 (4), 325-331 (1993).</li><li>Xiao, L., McCann, J. V., Dudley, A. C. <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+culture+expansion+of+tumor-specific+endothelial+cells.">Isolation and culture expansion of tumor-specific endothelial cells.</a> Journal of Visualized Experiments. (105), e53072 (2015).</li><li>Berry, R., Rodeheffer, M. S., Rosen, C. J., Horowitz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+residing+progenitors+(adipocyte+lineage+progenitors+and+adipose+derived+stem+cells+(ADSC).">Adipose tissue residing progenitors (adipocyte lineage progenitors and adipose derived stem cells (ADSC).</a> Current Molecular Biology Reports. 1 (3), 101-109 (2015).</li><li>Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+endothelial+progenitor+cells+from+adipose+tissue+as+potential+angiogenic+cell+sources+for+bladder+angiogenesis.">In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis.</a> PLoS One. 10 (2), e0117644 (2015).</li><li>Curat, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+blood+monocytes+to+adipose+tissue-resident+macrophages:+induction+of+diapedesis+by+human+mature+adipocytes.">From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes.</a> Diabetes. 53 (5), 1285-1292 (2004).</li><li>Sidney, L. E., Branch, M. J., Dunphy, S. E., Dua, H. S., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+evidence+for+CD34+as+a+common+marker+for+diverse+progenitors.">Concise review: evidence for CD34 as a common marker for diverse progenitors.</a> Stem Cells. 32 (6), 1380-1389 (2014).</li><li>Traktuev, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+population+of+multipotent+CD34-positive+adipose+stromal+cells+share+pericyte+and+mesenchymal+surface+markers,+reside+in+a+periendothelial+location,+and+stabilize+endothelial+networks.">A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks.</a> Circulation Research. 102 (1), 77-85 (2008).</li><li>Frontini, A., Giordano, A., Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+of+adipose+tissues:+a+niche+of+adipogenesis.">Endothelial cells of adipose tissues: a niche of adipogenesis.</a> Cell Cycle. 11 (15), 2765-2766 (2012).</li><li>Sengenes, C., Miranville, A., Lolmede, K., Curat, C. A., Bouloumie, 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+endothelial+cells+in+inflamed+adipose+tissue.">The role of endothelial cells in inflamed adipose tissue.</a> Journal of Internal Medicine. 262 (4), 415-421 (2007).</li><li>Ong, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+specific+cell-surface+markers+of+adipose-derived+stem+cells+from+subcutaneous+and+visceral+fat+depots.">Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots.</a> Stem Cell Reports. 2 (2), 171-179 (2014).</li><li>Ong, W. K., Sugii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+fatty+potentials+for+therapy.">Adipose-derived stem cells: fatty potentials for therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (6), 1083-1086 (2013).</li><li>Tchkonia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+two+human+preadipocyte+subtypes+with+distinct+capacities+for+replication,+adipogenesis,+and+apoptosis+varies+among+fat+depots.">Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.</a> American Journal of Physiology-Endocrinoloy and Metabolism. 288 (1), E267-E277 (2005).</li><li>van de Vyver, M., Andrag, E., Cockburn, I. L., Ferris, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinedione-induced+lipid+droplet+formation+during+osteogenic+differentiation.">Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.</a> Journal of Endocrinology. 223 (2), 119-132 (2014).</li></ol>

著者が明らかに何もありません。

本稿の焦点は、分離、拡張および CD31 cd34 陽性 + + ひと脂肪組織内臓や皮下のデポから血管内皮細胞の脂肪分化誘導方法論を提供することです。
齧歯動物または CD31 抗体、蛍光ラベルまたは磁気ビーズ18,に結合を使用して主にテクニックを使って人間の様々 な血管ベッドから血管内皮細胞の隔離のための方法論が報告されています。<sup class="xref...

最近の証拠は、マウスとヒトでは、白またはベージュ/ブラウン脂肪1,2,3に脂肪組織の血管に存在するセルのサブセットを区別できますを示しています。このような細胞の表現型の中間表現型4,5,6、7のスペクトルや平滑筋/周皮細胞、血管内皮細胞を支持する証拠との論争の主題であります。この方法論の開発の範囲は、CD31 cd34 陽性 + + 肥満人間から別の脂肪質のターミナルから分離した内皮細胞の脂肪分化能をテストするためだった。文献の他の研究は、潜在的な合計の間質血管の分数または知られている脂肪細胞の前駆細胞の2,8,9の脂肪細胞に着目します。現在既存の技術は、薬配信10特に脂肪組織の血管内皮細胞をターゲットすることができますので、このような細胞の脂肪分化を受ける可能性を理解白またはベージュの脂肪細胞へ誘導重要です。将来の目標とされた療法。
別のグループは、ひと脂肪組織11,12,13から血管内皮細胞を分離するサロゲートとして CD31 と CD34 マーカーの組み合わせを報告しました。通常、2 つの連続した手順と磁気ビーズを用いた正の選択を使用して分離が実行されます。本報告では、CD31 プラスチック ビーズと組み合わせて CD34 陽性磁気ビーズを用いた immunoseparation に利用されました。典型的な石畳の内皮形態の保全に関して逐次磁気 immunoseparation に優れたこの手法がわかった。また、脂肪の 1-2 g ほどから始まって拡大と脂肪細胞の誘導に必要な十分な細胞を生成することができました。皮下脂肪の小さなサンプル生検は、下流用電池の必要な量を生成するのに十分です。この面は可能性のある重要な特に場合は、このメソッドは人間の脂肪分化誘導に対する反応性のスクリーニングに使用されます。
文献で報告された他のシステムとは異なり、この方法は CD31 cd34 陽性 + + 細胞の脂肪分化誘導のための 2 つの成分を利用: ppar γ アゴニスト-ロシグリタゾン — とヒトインスリンです。重要なは、インスリンの使用量は、人間14post-absorptive インスリンを循環通常/高の範囲内にあります。カクテルの誘導に対応する能力と、細胞の in vitro、Akt のリン酸化による測定のインスリンに対する反応性の程度は相関しなかった。興味深いことに、この誘導のカクテルや実験条件を使用して、白とベージュ/ブラウン細胞のミックスは、サイズと分子マーカーの発現と細胞内脂質液滴の数によって決定された得られました。(白とベージュ)、細胞の表現型の定量的評価と一緒にこの簡単かつコスト効果の高い誘導プロトコルは、差別化されたベージュのバランスを変更することができます潜在的エージェントのスクリーニング: 白色脂肪細胞です。
このメソッドは、ひと脂肪組織の血管内皮前駆細胞の脂肪細胞形成の基になるメカニズムを理解するためトランスレーショナル アプローチを提供します。この特定の分離/差別化手法を使用して、捜査官は様々 な経路の細く、肥満の人間の様々 な脂肪質のターミナルから血管内皮細胞のサブセットの脂肪細胞の形成を担当に問い合わせることができます。

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

肥満は脂肪細胞の肥大によって主に脂肪組織の大規模な改造を伴います。極端な脂肪細胞成長は、インスリン、ローカル低酸素症および炎症に悪い応答が返される前駆細胞から機能的な白い脂肪細胞の分化を刺激し、脂肪細胞の人口の根本的な肥大を防ぐことができるしの削減とともにその結果、脂肪代謝の健康を改善できます。炎症。また、ベージュ/ブラウンの脂肪細胞の分化を刺激しと、全身のエネルギー消費量を増やすことができる減量、その結果します。このアプローチは、肥満 2 型糖尿病と心血管疾患などの併存の開発を防ぐことができます。
本稿では、分離、拡張、および共同 CD31 と CD34 のマーカーを表現するひと脂肪組織の血管内皮細胞のサブセットから白とベージュの脂肪細胞の分化をについて説明します。メソッドは、比較的安価ですし、労働集約的ではないです。人間の脂肪組織へのアクセスを必要として皮下のデポはサンプリングに適しています。このプロトコルの新鮮な脂肪組織は、病的肥満者からサンプル [ボディマス指数 (BMI) > 35] 肥満手術の手順中に収集されます。間質血管の一部から順次 immunoseparation を使用して、十分な細胞は、脂肪の 2-3 g ほどから生成されます。これらの細胞文化 10-14 日間で拡大することがでくことができますが, 凍結保存、通路 5-6 まで継と、脂肪細胞のプロパティを保持します。細胞は、脂肪細胞の ppar γ アゴニスト ロシグリタゾンとヒトインスリンの組み合わせを使ってカクテルを 14 日間扱われます。
この方法は脂肪の血管内皮細胞、脂肪細胞応答を駆動する分子メカニズムに関する実験についての証明書を取得するため使用ことができますまたは、脂肪細胞を強化することができます新しい薬物をスクリーニングするため応答指示ホワイトのどちらかベージュ/ブラウン脂肪細胞分化。小さな皮下のバイオプシーを使用して、この手法を使用して肥満と併存の治療のためベージュ/茶色と白色脂肪細胞を刺激することを目的とする臨床試験のための非レスポンダー科目を画面できます。

私達のプロトコルは CD31 cd34 陽性 + + ひと脂肪組織別拠点から血管細胞の脂肪分化能を決定するためのアプローチを提供することを目的します。簡単なフローチャート図は、図 1 aに表示されます。CD34 の発現細胞の正の選択を使用して最初のステップ > 95 %cd34 陽性細胞新鮮単離細胞 (図 1 a) の人口で起因します。重要?...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

白とベージュの脂肪細胞脂肪組織血管前駆細胞からの分化は、肥満の代謝改善の可能性を負いません。CD31 cd34 陽性 + + 人間の脂肪から内皮細胞分離および後続の in vitro拡張と白とベージュの脂肪細胞への分化のプロトコルについて述べる。いくつかのダウン ストリーム アプリケーションを説明します。

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

isolation-expansion-adipogenic-induction-cd34-cd31-endothelial-cells

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Immunology and Infection

11.7K ビュー.  Eastern Virginia Medical School. 白とベージュの脂肪細胞脂肪組織血管前駆細胞からの分化は、肥満の代謝改善の可能性を負いません。CD31 cd34 陽性 + + 人間の脂肪から内皮細胞分離および後続の in vitro拡張と白とベージュの脂肪細胞への分化のプロトコルについて述べる。いくつかのダウン ストリーム アプリケーションを説明します。

ビデオ: Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

免疫・感染学

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

の選択熱帯熱マラリア原虫</em人間の脳内皮細胞へのCytoadhesion用>寄生虫

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

ヒト母体·胎児のインターフェイスから白血球の単離

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

アデノウイルスに感染したヒト正常細胞からのウイルス複製コンパートメントに富むサブ核画分の単離

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

ラット精巣からのセルトリ細胞と尿細管周囲細胞の単離

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

ヒトおよびマウスの心筋標本からのマクロファージサブセットおよび間質細胞の分離

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...