私たちの技術的な助手にお世話になっております: u. Bröder、h. Shkarlet 実験手順の中で私たちを助けた。この作品は、部分的 (UniVax、契約番号 601738、TRANSVAC2、契約番号730964) EU 補助金及びヘルムホルツ協会助成金 (ハイ-IDR) によって賄われていた。資金源に影響しなかった研究デザイン、原稿や文書の提出する決定の世代。

本研究で使用されるすべてのマウスは、C57BL/6 背景からあった。すべての動物は、病原体フリーの条件の下で保たれました。(TierSchG BGBl ドイツの動物保護法の規範に従ってすべての実験が行われました。私 S 1105;25.05.1998) 許可番号 33.4-42502-04-13/1281 と 162280 の下で動物実験の倫理および州のオフィス (低いザクセン州事務所の消費者保護、食の安全) 低いザクセン州委員会によって承認されました。
1. CFSE 染色 OT-私は T 細胞と養子の転送
注: OT-私マウス固定 α と β 鎖一緒に OVA の免疫支配的なペプチドを認識する T 細胞受容体 (TCR) を表す transgenically 生成された動物は、SIINFEKL10,11。その結果、これらのマウスはかなり高い SIINFEKL 固有 CD8 数を持っている+ T 細胞 (97%)12通常または OVA 接種マウス (≤ 1%)13と比較した場合。
<ol><li>トレーサブルな CD8 の分離 OT から+ T 細胞-私 T リンパ球特定Thy1 (Thy1.1) の対立遺伝子を発現するマウス。<ol>
<li>6-9 週齢の OT を安楽死させる-私 CO2吸入によるマウスに続いて頚部転位14。手術ペンチや鉗子の助けを借りて体から皮膚を切り離す手術のハサミで皮膚を切断することによって脾臓および主要なリンパ節 (鼠径部、腋窩、頸部ペアのみ) を分析し、各を含むペトリ皿 (60 x 15 mm) に配置、100 μ m 孔メッシュ カップ組織研削やセル リリース。</li>
		<li>3-5 mL の完全 RPMI 培 (RPMI 1640 年 10 %v/v FCS、100 U/mL ペニシリン、ストレプトマイシン 50 μ G/ml) 氷で保たれるペトリ皿 (それぞれ 1 つのメッシュ カップ) で維持されます。</li>
		<li>シリンジのプランジャーまたは同様の滅菌器の助けを借りて、臓器をマッシュ (前の切断は必要ありません) 15 mL 遠沈管の結果単一細胞懸濁液を収集し、。</li>
		<li>4 ° C で 10 分間 300 x g で細胞懸濁液を遠心分離します。上清を捨てます。遠心分離で 10 mL の冷 PBS のセルを洗浄し、塩化アンモニウムのバッファー (ACK バッファー、市販) の 1 mL/脾臓にペレットを再懸濁による脾臓から赤血球を溶解させます。氷の上 1.5 分細胞をインキュベートし、その後 (前に、と) は遠心分離によって 10 ml の冷 PBS のセルを洗ってください。</li>
		<li>再 1 ml の PBS (pH 7.2) 含む 5% ウシ胎児血清磁気分離のための同じ管でペレットを中断します。</li>
	</ol>
</li>
	<li>磁気分離を実行する CD8 の負の淘汰に進みます+ T の製造元の指示に従って磁気分離キットを使用してセルまたはプロトコル15を公開します。</li>
	<li>CFSE 染色するには、まずカウント CD8 の数+ T OT から得られた細胞-私自動化された細胞カウンター (パーティクル カウンター) を使用してマウス。1-5 107セル/mL 37 ° C、15 ml の隼の管の光から保護で 7 分の 5 μ m CFSE PBS で 1-5 mL の量 x を染色します。</li>
	<li>牛胎児血清の同じボリューム (1:1) をセルに追加することによって CFSE 染色を癒やす、光から保護されている 37 ° C でさらに 7 分間インキュベートするそれら。10 ml の PBS のセルを 2 回洗います。</li>
	<li>自動化された細胞カウンターを使用してセルをカウントします。3-5 x の 100 μ L の 10 の6セル/マウスを静脈内に注入するために 3-5 x 107セル/mL の PBS のセル番号を設定 (静注) 尾静脈を介して。</li>
	<li>尾静脈注射を実行するには、適切な restrainers でマウスを固定します。背部区域と尾静脈血管拡張を許可する 1 〜 3 分のマウスから 20 〜 25 cm の間置かれた赤い光ランプを使用して注入するマウスの尾を温めます。 注: これは簡単静脈検出と細胞懸濁液の管理に役立ちます。</li>
	<li>(マウスとランプの間に置かれた手) でもマウスのホットし、尾静脈がはっきりと見える、インジェクションに進むではないです。25 ゲージ針161 mL 注射器を使用して水平または背の尾静脈にセルを挿入します。</li>
</ol>2. 予防接種 (補助 + 遠藤無料 OVA)
<ol><li>OT を移植されたマウスを置く-私は麻酔室で (ステップ 1.7、図 1) を細胞し、酸素のイソフルラン麻酔マシン14管理します。</li>
	<li>マウスを麻酔下で完全に眠っているとき、チャンバーから取り出すし、臀筋浅(外側腰部) にきれいな注入を行うために電気髪のナイフを使用して領域のマウスの毛皮を剃るアプリケーション領域の景色の良い。</li>
	<li>ワクチン、25 ゲージ針を使用して剃毛エリアでサウスカロライナの 50 μ L を注入します。</li>
</ol>3 フローサイトメトリー解析のための汚損およびリンパ球の分離
<ol><li>脾細胞およびリンパ球の分離<ol>
<li>頚部転位続いて CO2吸入ワクチン接種マウスを安楽死させます。</li>
		<li>排水、遠くのリンパ節や脾臓を抽出し、100 μ m 孔メッシュ カップ組織研削と細胞のリリースを含む別のペトリ皿に配置します。1.1.2 1.1.3 からの手順に従います。</li>
		<li>遠心分離後、上清をデカントし、再残骸 PBS の同じボリューム (約 100 μ L) の細胞ペレットを中断します。</li>
	</ol>
</li>
	<li>フローサイトメトリー解析のため染色<ol>
<li>15 mL 遠心管中の表 1に示されている濃度で染色抗体を含むマスター ミックスを調製、染色ミックスを集中してこのように 2 倍を降伏します。サンプルあたり 100 μ L を持っているマスター ミックスの十分な量を準備します。セルと 1:1 のマスターの組合せをミックスし、4 の ° c で 30 分間インキュベートします。 注: サンプルあたり最終染色巻 200 μ L (抗体のマスターの組合せの細胞ペレット 100 μ L の 100 μ L) をする必要があります。</li>
		<li>1.1.3、10 mL の PBS を 2 回追加するで説明するよう 2 回遠心分離によって細胞を洗ってください。</li>
		<li>0.5 - 1 染色細胞を再停止集録および流れの cytometer チューブへの転送停止用 mL の PBS。常に氷の上のサンプルを維持し、光から保護されています。</li>
	</ol>
</li>
</ol>4. フローサイトメトリー
<ol><li>常に 70-100 μ m のフィルターを使用してセルのサンプルがプレフィルターです。</li>
	<li>表 1 (ビーズまたは細胞) に詳細な fluorophores の単一の染色補正コントロールを準備します。 注: 補償する必要がありますの cytometer または分析ソフトウェア17,18,19で実行し、すべてのサンプルに適用されます。</li>
	<li>図 2にゲートの戦略に従います。前方散乱領域 (図 2 a)、もう一度広い側の散布面積 (SSA は、図 2 b) ダブレットを除外するために対側方散乱と前方散乱の高さで簡単にゲート集団。</li>
	<li>門真 CFSE を差別するために BV 650 (自動蛍光) FITC (CFSE) 対をプロットすることによって図 2 bから人口は高自動蛍光細胞から細胞を染色しました。ビーズや補正コントロールに BV 650 に共役抗体で染色のセルが含まれます。OT を門に FITC 対 BV 650 のこのプロット (図 2) を使用-私 CFSE は細胞を染色します。</li>
	<li>受信者マウス対ドナー由来細胞を区別するために Pe Cy7 (Thy1.1 - CD90.1-) を APC (CD8) 対の図 2 Cから人口をゲートします。 注: セルに由来する OT-私 WT (C57BL/6、受信者) マウス由来細胞は Thy1.2+ (CD90.2) (図 2 D) に対し、ドナーのマウスは Thy1.1+ (CD90.1)。いくつかの研究所がある OT-私 Thy1.2 バック グラウンドで WT (C57BL/6)、Thy1.1 のマウス。この場合は OT の Thy1.2 抗体を使用する必要が-ゲート携帯ですね。</li>
	<li>CD8 を再ゲート+細胞 Thy1.1+人口 APC (CD8) 対 BV 450 (CD4) で構成されるゲートにプロットして (図 2 e)。</li>
	<li>CD8 のヒストグラム表示を使用して、図2 e ゲート人口を表示+ CFSE (FITC、530/15 チャンネル - 青色レーザー-)、細胞増殖の人口門 10 から強度を含む2レベルまで、コントロール分裂していない人口は (強度 10 〜5、CFSE 染色と流れの cytometer の電圧設定の有効性に応じて) (図 2 f)。</li>
	<li>マーカーにする、(図 2 には表示されません) 付加ゲート プロット FITC 対 L/D (450/20 チャンネル、紫外レーザー) 増殖の評価に並列に死んだ細胞を評価するために。</li>
	<li>補正コントロールのイベントの少なくとも 5000 と流れの cytometer でサンプルから 10.000 イベント (図 2E、ゲート) を取得します。低または中程度の流れの cytometer を実行 (20.000 イベント/秒の流量を超えない)。</li>
</ol>

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C., 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=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, 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=Comparison+of+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., 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=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, 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=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

著者が明らかに何もありません。

現代のワクチンは、理想的にしまいこむ精製抗原とアジュバント、リポソーム、ウイルス様粒子、ナノ粒子やライブのベクトルのような配信システムの可能な追加です。ワクチンを設計するときの重要な側面は、臨床医学のニーズによると右の補助を選択することです。範囲の一部は好む細胞性免疫応答 (またはその両方) と体液性、全身性免疫反応 (またはその両方)、対ローカルの選挙とタ...

細胞傷害性 T リンパ球 (CTL) を誘導するワクチンは、がん1の特定の種類を戦うために開発されているキーの治療です。CTL はまた細胞内病原体2に対する予防ワクチンのため重要です。また、CTL は、初期の生活の感染症5と戦うための CTL はまた決まる人新生児3,4などリスク集団の機能的アクティブないくつかの免疫防御機構の 1 つです。この点では、CTL 応答 (ミョウバン) は発生しませんするアジュバント開発された呼吸器合胞体ウイルス (RSV) に対するワクチンは乳児6感染症時に重篤な合併症につながるワクチンの完全な失敗に終わった。予防接種のこれらの負の影響は CD8 によって反転できます+ T 細胞応答7。我々 は以前、インターフェロン遺伝子 (スティング) 作動薬のいくつかの刺激によって誘発される (タイプ I のインターフェロン) 主なサイトカインの増殖を測定することによって部分的にこれらのアジェバントの8、によって生成された CTL 応答のため不可欠であることを示しています。OT-T 細胞ワクチン接種と CTL 誘導機能の測定で観測されたこれらの結果を使用して拡張予防接種スケジュール9後。OT の拡散の測定-私は CD8+ T 細胞の野生型 (WT) C57BL/6 受信者マウス carboxyfluorescein 染色エステル (CFSE) 色素希釈が生成するワクチンのアジュバントの機能の推定SIINFEKL (OVA 卵白アルブミンの免疫支配的なペプチド) のクロス プライミング。この手法のバリエーションが広く OT の増殖の評価-私は CD8+と OT II CD4+ T 細胞。たとえば、それはいない選択したサイトカイン (KO マウス)、あるいは WT 動物抗原リコール後ワクチンの有効性を測定するために使用されています。CFSE 染色 OT の受身伝達後、短いプロトコル (4 日間実験) を考案-私は CD8+ T 細胞、50 の 1 つの線量から成る皮下皮下接種試験アジュバントを添加したエンドトキシン OVA の μ g を投与(図 1)。フォロー アップの結果ワクチン接種後 48 時間は、CTL 応答を生成するアジュバントの能力の信頼性の高い証拠を提供します。この戦略によって脾臓 (または遠隔リンパ節) に CTL 活性を測定することによって応答の範囲と同様、予防接種後排出リンパ節における局所免疫応答の効力を評価することが可能です。

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

モダンなサブユニット ワクチンの評価では、中和抗体の生成は重要ですがアジュバントの選択は不十分であることを明らかにします。したがって、細胞傷害性 T リンパ球 (CTL) の反応を促進することができる体液性および細胞免疫促進機能を備えたアジュバントが急務します。したがって、忠実な監視クロスプライミングを誘導し、その CTL 世代を強化補助候補者のワクチンの開発の重要なステップを表します。ここで提案する SIINFEKL 固有を使用するメソッドのアプリケーション (OT-私) T 細胞モデル抗原卵白アルブミン (OVA) は、生体内で異なる補助候補の存在下でのクロス プレゼンテーションを監視します。このメソッドは、最高のクロス プライミング機能を備えたアジュバントを選択する急速なテストを表します。CD8 の増殖+ T 細胞クロスプライミングの最も貴重な徴候、また、アジュバント クロス プレゼンテーションの相互関係として考えています。この機能は、リンパ節や脾臓のようなさまざまな免疫器官で評価できます。CTL 世代の範囲も監視できます、それによりローカルの性質に洞察を与えて (主にリンパ節をドレイン) または全身性反応 (遠くのリンパ節や脾臓)。さらにこの手法も従来型と遺伝子組み換えマウスの系統に使用される可能性を提供していますクロス プレゼンテーションの特定の経路を阻害することができます薬をテストするための複数の変更をことができます。要約すると、ここで紹介するアプリケーションは業界や学界でワクチンの研究所で、開発または化学のアジュバント ワクチンの研究と開発のための変更のため役に立つでしょう。

CTL の発電容量をに対して転送 OT の増殖を測定することによって評価我々 異なるアジュバント (その ADJ1 および ADJ2) の組み合わせを使用して治療法をテストするために-私は CD8+ T 細胞のフローサイトメトリー (図 2)。このため、以前ドレイン リンパ節および脾臓 (表 1) から分離された細胞を染色しました。CD8 の増殖を測定?...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

標準的な免疫学的技術のアプリケーションをご紹介 (CFSE 染色 OT-私増殖) 急速に補助を介した細胞傷害性 T リンパ球 (CTL) の生成をモニターするつもり体内。CTL 容量の高速推定、がん治療用ワクチンと同様に、細胞内の病原体に対する予防ワクチンの開発に役立つ。

急速なIn Vivo評価補助の細胞傷害性 T リンパ球生成機能のワクチン開発のため

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K ビュー.  Helmholtz Centre for Infection Research. 標準的な免疫学的技術のアプリケーションをご紹介 (CFSE 染色 OT-私増殖) 急速に補助を介した細胞傷害性 T リンパ球 (CTL) の生成をモニターするつもり体内。CTL 容量の高速推定、がん治療用ワクチンと同様に、細胞内の病原体に対する予防ワクチンの開発に役立つ。

ビデオ: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

精巣カプセル内奇形腫の発生

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

全血におけるエノキサパリン抗凝固効果の迅速なポイントオブケアアッセイ

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

骨肉腫細胞における光線力学療法の細胞毒性有効性<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

次世代シーケンシングのための収集と唾液DNAの抽出

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

マイクロコンピュータ断層撮影とヌードマウスへのヒト脂肪注入の生存率の評価

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

急速なIn Vivo評価補助の細胞傷害性 T リンパ球生成機能のワクチン開発のため

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