著者は、ナントから大学病院動物施設 (UTE) ナントの畜産とケア、細胞と組織イメージング、イメージング ナント大学 (MicroPICell) の中核施設とフローサイトメトリー施設 (Cytocell) のスタッフに感謝します。彼らの専門家のテクニカル サポート。この仕事 INSERM、CNRS、ユニヴェルシテ ・ ド ・ ナント、Institut 国立デュがん (インカ #PLBio2014-155)、リーグのナシオナル contre le がん (AO 地域間 2017 年)、欧州コンソーシアム時代ネット Transcan2 によって資金が供給された (Immunoglio)。チームは財団注ぐラ凝った Medicale (DEQ20170839118) によって資金を供給します。LabEX 囲碁のコンテキストでこの作品を実現し、IHU セスタス プログラムでサポートされている、国家研究機関 Investissements d'Avenirを介してプログラム ANR-11-LABX-0016-01 と ANR-10-IBHU-005、それぞれ。IHU セスタス プロジェクトはナント メトロポールとペイ ・ ド ・ ラ ・ ロワール地域でもサポートされます。著者は、Chirine ラフィア ・が原稿を修正ヘルプを提供することをありがちましょう。

次プロシージャを含む動物科目制度ガイドライン (契約 #00186.02; ペイ ド ラ ロワール [フランス] の地域倫理委員会) によるとを行った。人間の PBMCs は情報に基づいた健康なドナー (エタブリセモン Français デュ歌ったナント, フランス) から集められた血から分離しました。すべての手順は、無菌条件の下で実行されます。
1. 非特異的細胞エフェクター T 細胞の拡張
<ol><li>準備し、フィーダー細胞 35 Gy を照射します。2 x 105 - 4 x 105エフェクター細胞の刺激の 3 つの明確な健康なドナーから 10 x 106 PBMCs と 1 x 10 の6 BLCLs を準備します。</li>
	<li>フィーダー細胞と 15 ml の 10% の熱不活化 FCS、2 ミリメートル L グルタミン、ペニシリン (100 IU/mL) とストレプトマイシン (0.1 μ g/mL) と 300 IU/mL 組換え IL-2 添加 RPMI のエフェクター細胞を再懸濁します。</li>
	<li>最終濃度 1 μ G/ml に PHA L を追加、慎重にミックス、および 150 μ L/ウェルの 96 穴 U 底プレート細胞懸濁液を配布します。</li>
	<li>37 ° C で、加湿雰囲気で 5% CO2で孵化させなさい。</li>
	<li>大細胞塊文化井戸のフォームまでプレートの日常点検 (~ 7 日)。</li>
	<li>1 x 106セル/mL の新鮮な培養液中で培養用フラスコにセルを転送します。</li>
	<li>(2 倍の週) を数えることで細胞数を決定し、新鮮な培地の 1 x 106セル/mL でそれらを維持します。 注: エフェクター免疫細胞は静止状態 (初期増幅刺激後通常 3 週間) で治療的投与の準備ができてする必要があります。純度とこれらのエフェクター細胞の反応性は、生体内注入 (例えば、生体外の試金と) 前にチェックしなければなりません。</li>
</ol>2. 術前のエフェクター細胞の準備
<ol><li>エフェクター細胞数を確認した後遠心分離 (300 x gで 5 分) で 50 mL のチューブでエフェクター細胞を収集します。 注: 任意の損失を補うために細胞 (例えば50 × 106) の超過分を準備します。</li>
	<li>慎重に上澄みを除去し、15 mL の滅菌 PBS と 300 × gで 5 分間遠心洗浄を実行するのに細胞を再懸濁します。</li>
	<li>慎重にそして完全に上澄みを除去し、1 mL の滅菌 PBS で細胞ペレットを再懸濁します。</li>
	<li>5 分間 300 × gで遠心分離を 1.5 mL マイクロ チューブの再懸濁のセルを転送します。</li>
	<li>慎重にそして完全にゆっくりとピペッティング上澄みを削除します。</li>
	<li>マウスごと滅菌 PBS の 8 μ L で細胞ペレットを再懸濁します。 注: これは重要なステップです。</li>
	<li>マイクロ ピペットを用いて細胞懸濁液の量を測定します。必要に応じて、滅菌 PBS (20 × 106線量あたり 15 μ L の PBS セル) を追加し、慎重に混ぜます。</li>
	<li>15 と 20 μ L (強制的 < 20 μ L) 間マウスあたり最終巻にあるマイクロ ピペットを使用して確認してください。</li>
	<li>定位注射まで氷の上細胞を保ちます。 注: 3 時間以上縦長はテストしませんでした。</li>
</ol>3. 定位注射
<ol><li>
機器のセットアップ
	<ol>
<li>組み立てるし、頭蓋内注射 (例えば、シリンジ サイズ、音量、注入の速度) の精度を確保するための製造元の指示に従って小動物定位脳手術フレームをキャリブレーションします。 注: 低速注入率は (すなわち2 〜 3 μ L/分) 推奨します。</li>
		<li>感染症から微生物安全キャビネット (MSC) 楽器の無菌性を維持し、マウスを保護するための下で材料をインストールします。 注: 場所"等温ブロック」の 37 ° C の水浴中このシステムは、手術中にマウスの体温を制限します。手続き後のケアのために必要であるパッドを加熱は、連続温度監視中に使用する必要があります。</li>
	</ol>
</li>
	<li>
動物の術前準備
	<ol>
<li>ケタミン (10 mg/mL) とマウスの体重の 10 μ L/g でキシラジン (0.1 mg/mL) の腹腔内注入マウスを麻酔します。</li>
		<li>完全な麻酔と動物の鎮痛を確保するつま先ピンチ テストを実行します。 注: 任意の動きが非深い鎮痛の表示、操作を繰り返す前に、数分以上が必要な場合は。</li>
		<li>マウスは麻酔は正しく、(間に 2 つの耳、鼻まで) 手術部位から毛皮を削除するのにはさみを使用します。</li>
	</ol>
</li>
	<li>
術前セル準備
	<ol>
<li>任意のセルの凝集を防ぐために各注入前にピペットで細胞の (複数回) を慎重に再懸濁します。</li>
		<li>慎重に泡の吸引を避けるために 22 G 変らずに必要な細胞懸濁液容積 (15-20 μ L) を描画します。 注: 変らずにこのセルの読み込み手順は注入されたボリュームでの差異を最小化することが重要です。各コホートのエフェクター細胞学も数を確保するための手続任意のセルの凝集を防ぐために個別注射用セルを再読み込みします。</li>
		<li>その後、適応シリンジ ポンプに注射器を配置します。</li>
	</ol>
</li>
	<li>
手続きの注意
	<ol>
<li>ポビドン ヨードの 5% 溶液を少なくとも 3 に浸した綿棒で手術部位を消毒 x。</li>
		<li>マウスの目の角膜の乾燥防止のために潤滑の眼軟膏を配置します。</li>
		<li>暖かい等温ブロック上の定位脳手術フレームに麻酔下のマウスの位置は、手術中にマウスの温度を維持し、死亡率を制限する滅菌ラップで覆われて。 マウスの鼻と歯注: はプロシージャの間に十分な呼吸流量を確保するため、歯のバーの上適切に配置する必要があります。</li>
		<li>歯のバーの上にマウスが、一度頭を固定するマウスの耳の下でしっかりとイヤバーを締めます。 注: するまたは呼吸の妥協に鼓膜を損傷しないように注意してください。</li>
		<li>頭蓋骨を公開する後方前方から頭蓋の上部に沿って滅菌ハサミで 1-2 cm 正中矢状皮膚切開を確認します。</li>
		<li>矢状面と冠状縫合糸 (図 1に示すように) 注入前に定位のローカリゼーションのためのランドマークとして機能する (前) 交差点を識別します。</li>
		<li>このポイントより上, マイクロシリンジを配置します。</li>
		<li>変らず右横 2 mm と 0.5 mm 前の前方に移動します。</li>
		<li>あらかじめ定義された座標で滅菌ドリルで頭蓋骨に小さな穴を作り、ドリルを使用する。脳の任意の外傷性の損傷を避けるために表在性のままに注意してください。 注: このプロトコルでは、免疫細胞が確立された腫瘍 (腫瘍細胞の注入後 1 週間) 内で注入されました。皮膚がリニューアル オープン (瘢痕)、注射をする必要があります (穴は注入後 2 週間までまだ存在一般に) 腫瘍細胞移植に使用される同じ座標で行われます。座標は、脳実質13,14の腫瘍とエフェクター細胞を注入するために選ばれました。</li>
	</ol>
</li>
	<li>
免疫エフェクター細胞の注入
	<ol>
<li>慎重に穴に、変らずを挿入し、ゆっくり動いて、硬膜下 3 mm とエフェクター細胞を注入する前に 2.5 mm の最終的な深さにして後方 0.5 mm の針を転送します。</li>
		<li>2-3 μ L/分でエフェクター細胞の注入を実行し、射出時間に沿ってマウスを監視します。</li>
		<li>注入が完了すると、わずか 1 mm の針を撤回し、変らずを注入サイトから漏洩を防ぐため、変らず撤退完全にゆっくりと前に 1 つの追加分の場所に保管します。 注: 次の定位脳手術装置から動物の除去、今後実験用噴射装置がすぐにきれい。</li>
	</ol>
</li>
	<li>
手術後のケアとマウスのフォロー アップ
	<ol>
<li>定位脳手術フレームから動物を削除してすぐに切開部位のポビドン ヨード溶液の 5% を適用し、適切な外科的縫合糸で皮膚を閉じます。</li>
		<li>傷口に直接 2% リドカイン ゲルを適用し、手続き後の鎮痛のため皮下注射ブプレノルフィンの 0.15 μ g/g を管理します。</li>
		<li>場所戻ってマウスの適切な体温を維持し、低体温症を避けるために、加熱パッドの上のケージに麻酔下のマウスは 37 ° C に設定。 注: 別のハウジングは必要ではありません。</li>
		<li>それは完全に麻酔から回復するまでマウスを監視し、ハウジング ルームにそれを転送します。 注: までに、このプロトコルもサポートいくつかの予期せぬ合併症が発生したとして (注入されたマウスの < 5%)。</li>
		<li>毎日、マウスを監視し、(例えば、背を丸めて姿勢、減らされた移動性、虚脱、または重要な体重減少 [≥ 15%])、健康の減少兆候が観察されるときそれらを安楽死させます。</li>
	</ol>
</li>
</ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+responses+of+the+central+nervous+system.">Innate and adaptive immune responses of the central nervous system.</a> Critical Reviews in Immunology. 26 (2), 149-188 (2006).</li><li>Louveau, 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=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. 523 (7560), 337-341 (2015).</li><li>Menei, P., 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=Local+and+sustained+delivery+of+5-fluorouracil+from+biodegradable+microspheres+for+the+radiosensitization+of+glioblastoma:+a+pilot+study.">Local and sustained delivery of 5-fluorouracil from biodegradable microspheres for the radiosensitization of glioblastoma: a pilot study.</a> Cancer. 86 (2), 325-330 (1999).</li><li>Menei, P., 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=Stereotaxic+implantation+of+5-fluorouracil-releasing+microspheres+in+malignant+glioma.">Stereotaxic implantation of 5-fluorouracil-releasing microspheres in malignant glioma.</a> Cancer. 100 (2), 405-410 (2004).</li><li>Salot, S., 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=Large+scale+expansion+of+gamma+9+delta+2+T+lymphocytes:+Innacell+gamma+delta+cell+therapy+product.">Large scale expansion of gamma 9 delta 2 T lymphocytes: Innacell gamma delta cell therapy product.</a> Journal of Immunological Methods. 326 (1-2), 63-75 (2007).</li><li>Bennouna, J., 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=Phase-I+study+of+Innacell+gammadelta,+an+autologous+cell-therapy+product+highly+enriched+in+gamma9delta2+T+lymphocytes,+in+combination+with+IL-2,+in+patients+with+metastatic+renal+cell+carcinoma.">Phase-I study of Innacell gammadelta, an autologous cell-therapy product highly enriched in gamma9delta2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma.</a> Cancer Immunology, Immunotherapy. 57 (11), 1599-1609 (2008).</li><li>Singh, S. 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+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li></ol>

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養子伝選択ネイティブまたは設計された免疫エフェクター細胞腫瘍浸潤の脳腫瘍、非変形細胞15、に対して反応性を制限することの世話などを効率的に治療するために有望なアプローチを表す 16,17,18。ただし、脳を含む中枢神経系は、血液脳関門の存在と古典的なリンパ排液システム<sup class="xref"...

糸球体基底膜 (WHO グレード IV 星状細胞腫) は、成人では最も頻繁に、積極的な脳腫瘍の症例です。積極的な治療法は手術とラジオ化学療法を組み合わせた、GBM 変わりません非常に予後不良 (14.6 カ月と 2 年死亡 > 73% の生存期間中央値)1に関連付けられています。これは、いくつかの効率的な治療の進歩が2の最後の 10 年を検証されている証拠します。効果的な治療戦略3,4、5のデザイン候補の中で免疫療法6現在検討するいると高度侵襲とラジオ/化学療法抵抗性の腫瘍を削除セルは、迅速かつ致命的な腫瘍再発7への重要な貢献の疑いがあります。様々 な潜在的な免疫学的ターゲットが識別されたとの提案された免疫療法や自然を含むまたは変更した αβ GBM の腫瘍抗原など応力誘起分子8,9、ϒδ T リンパ球 10。選択した糸球体基底膜反応免疫細胞エフェクターを管理する可能性は、残存腫瘍のサイトに直接エフェクター リンパ球の上昇量を提供するユニークな機会を表します。この戦略をサポートするため、我々 は最近脳腫瘍 GBM 患者9,11の開発を忠実に同所性同種ヒト糸球体基底膜移植を運ぶ免疫不全マウスに基づくモデルに要約を示しています。さらに、これらのモデルに対して転送された同種の人間 Vϒ9Vδ2T のリンパ球の強い抗腫瘍活性効率を示すため使用されました。
このプロトコルでは、糸球体基底膜、同種リンパ球の養子の転送に基づくなどの脳腫瘍の定位放射線免疫療法を達成するため重要な実験手順について説明します。記事ショー: (i) 治療同種免疫エフェクター T リンパ球, リンパ球 Vϒ9Vδ2T 人間; などの増幅(ii) これらのエフェクター T のリンパ球の注射用の準備(iii) 内腫瘍近傍の脳定位放射線管理のための手順。この記事は、定位注射後これらの細胞のエフェクターの行動に洞察力を提供します。
ここで紹介する治療法は、それぞれの脳腫瘍免疫不全マウスの線量あたり20 x 106エフェクター細胞の注入に基づいています。初期の in vitro拡張ステップは、免疫細胞の大量生産に必要です。したがって、非特異的細胞拡張フィトヘマグルチニン (PHA L) を使用して実行され、同種フィーダー細胞を照射: 健常者とエプスタイン バール ウイルス EBV 変換 B リンパ芽球細胞から末梢血単核球 (PBMCs)EBV を含む培養 1 μ G/ml シクロスポリン A の存在下で、マーモセット B95 8 細胞ラインから上清中の体外感染による PBMCs 由来株 (BLCLs)
抗糸球体基底膜の免疫細胞が比較され、生体外の試金9から選択。これらのエフェクター細胞がアクティブになります、標準プロトコルを使用して (例えば、人間 Vγ9Vδ2 T リンパ球9またはヒト抗ヘルペス ウイルス T リンパ球12) 性質に従って最低純度として日常的に、> 80% で増幅フローサイトメトリー解析をチェックします。下記のとおりセル拡張プロシージャは、様々 な人間のリンパ球のサブセットに適用されます。

<table border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBMCs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:232px;">from 3 different healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BLCLs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td>from 3 different donors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Roswell Park Memorial Institute medium (RPMI)</td>
			<td style="width:136px;">Gibco</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">FCS</td>
			<td style="width:136px;">Dutscher</td>
			<td style="width:119px;">S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-glutamine</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">penicillin/streptomycin</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">IL-2</td>
			<td style="width:136px;">Novartis</td>
			<td style="width:119px;">proleukin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PHA-L</td>
			<td style="width:136px;">Sigma</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Stereotaxic frame</td>
			<td>Stoelting Co.</td>
			<td align="right">51600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mouse adaptator for stereotaxic frame&#160;&#160;</td>
			<td>Stoelting Co.</td>
			<td align="right">51624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microsyringe pump injector&#160;</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">UMP3-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil Syringe</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NSG mice</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:119px;">NSGSSFE07S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ketamine</td>
			<td style="width:136px;">Merial</td>
			<td style="width:119px;">Imalg&#232;ne 1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylazine</td>
			<td style="width:136px;">Bayer</td>
			<td style="width:119px;">Rompur 2%</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Scissors</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">201758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Forceps</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">501215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OmniDrill 115/230V</td>
			<td style="width:136px;">WPI</td>
			<td align="right">503598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Vicryl 4-0</td>
			<td style="width:136px;">Ethicon</td>
			<td>VCP397H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylocaine</td>
			<td style="width:136px;">Astrazeneca</td>
			<td>3634461</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

膠 (GBM)、成人では、最も頻繁に、積極的な脳腫瘍の症例は、予後不良な一般的に関連付けられている、最後の 10 年間で不足している効率的な治療法が提案されています。新しい治療戦略を設計するための有望な候補者、間細胞免疫療法は高度侵襲を排除する対象となっているし、化学療法放射線腫瘍細胞、可能性がありますこの癌の迅速かつ致命的な再発に関与します。したがって、腫瘍近傍の人間の Vϒ9Vδ2 の T リンパ球など、同種の糸球体基底膜反応免疫細胞エフェクター administration(s) 効率を提供するユニークな機会を表しますが、非常に直接の治療薬に集中して、脳の悪性腫瘍のサイト。同所性同種人間原発性脳腫瘍を運ぶ免疫不全マウスにおける準備と同種の人間のリンパ球の脳定位固定装置の管理のためのプロトコルを紹介します。本研究は、可能性と intrabrain 腫瘍ベッド内で同種の人間のリンパ球の定位注射に依存するこれらの細胞免疫療法の抗腫瘍効果の前臨床証拠の概念を提供します。

本研究では、脳内定位注射腫瘍ベッド内で直接実行に基づく腫瘍を持つマウスの細胞免疫エフェクター細胞の養子の転送の戦略について説明します。
大規模な注入量に関連付けられている脳損傷のすべてのリスクを最小限に抑える、エフェクター細胞懸濁液 (20 x 106 15 20 μ L の PBS セル) を集中する必要があります?...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

同所性同種人間神経膠腫異種移植片のマウスモデルの細胞傷害性免疫細胞の定位の養子転送

同所性同種人間原発性脳腫瘍を運ぶ免疫不全マウスにおける準備と同種の人間のリンパ球の脳定位固定装置の管理のためのプロトコルを紹介します。本研究は、実現可能性と intrabrain 配信細胞免疫療法の抗腫瘍効果の概念実証を提供します。

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

stereotactic-adoptive-transfer-cytotoxic-immune-cells-murine-models

Research

JoVE Journal

Immunology and Infection

7.8K ビュー.  CRCINA. 同所性同種人間原発性脳腫瘍を運ぶ免疫不全マウスにおける準備と同種の人間のリンパ球の脳定位固定装置の管理のためのプロトコルを紹介します。本研究は、実現可能性と intrabrain 配信細胞免疫療法の抗腫瘍効果の概念実証を提供します。

ビデオ: Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

実験的転移とCTLの養子移入免疫療法のマウスモデル

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

糖尿病CD4の養子移入+ T細胞によるマウスの加速された1型糖尿病の誘導

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

ケアの膠芽腫の標準のコンテキストにおける養子療法のためのCAR T細胞の生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

細胞毒性の評価と肺癌精密カット スライスで物質の免疫調節効果

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.