This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

施設内倫理委員会によって承認された患者のインフォームドコンセントは、ヒト細胞の使用を取得する必要があります。 ヒト末梢血単核細胞（PBMC）の1の密度勾配分離<ol><li> 25ミリリットルのピペットで50mlチューブに全血をヘパリン化25ミリリットルを追加します。 <ol><li>リン酸緩衝生理食塩水（PBS）25mlで細胞を希釈します。 </li></ol></li><li>新しい50mlのチューブにフィコール - パック密度勾配の15ミリリットルを追加し、チューブを傾けながら、全血のない混合を伴うがないように、非常にゆっくりと慎重に、密度勾配の上に希釈した細胞懸濁液の25ミリリットルを追加密度勾配、 すなわち 、血液の密度勾配界面の乱れ。 </li><li>遠心分離（20℃）ブレーキなし温度制御スイングバケットローターで30〜40分間グラム×400で、ステップ1.2からサスペンション。 注：3つの異なる層を遠心分離した後に明らかであるべきである：目プラズマであること電子上層;下部透明層は、フィコール - パーク密度勾配です。薄い、中間の細胞層はPBMCをさ</li><li>吸引し、単核細胞 （すなわち、リンパ球、単球、および血小板）の間期層を乱さないように注意して、パスツールピペットで吸引することにより、上層を捨てます。 </li><li>慎重に新しい50mlの遠心管に10 mlピペットでこの単核細胞層を収集します。 </li><li> 20mlのPBSでチューブを埋める、よく混ぜ、20℃で10分間、300×gで遠心します。遠心分離後、上清を完全に削除します。 </li><li> 20℃で10〜15分間200×gで細胞をPBS 20ml中のペレットと遠心機を再懸濁します。遠心分離後、上清を完全に削除します。 注：このステップは、血小板-不要内のPBMCですが削除されます。このステップは、血小板の完全な除去を確実にするために繰り返すことができます。 </li><li>特定の集団のない選択がされていない場合1mlのを停止するセル数16を実行した後の培養のための所望の白血球完全培地中、再懸濁細胞ペレット（10％ウシ胎児血清（FBS）、1％L-グルタミン、およびRPMI-1640培地中に1％のペニシリン/ストレプトマイシン）次のステップに進む前に、10 7のPBMCあたりの媒体。 </li></ol>白血球集団の2磁気標識（特定の集団の選択が必須でない場合は、ステップ4に進んでください） <ol><li>完全に10分と上清を除去し、300×gで遠心分離PBMC懸濁液。 </li><li> 10 7全細胞あたりのPBS80μlの再懸濁細胞ペレット。 </li><li> 10 7全細胞あたりの指定された磁気ビーズを20μl（単球を単離するための、すなわち、CD14磁気ビーズ、Tリンパ球の単離のためのCD4磁気ビーズ）を追加します。 </li><li>よく混合し、冷蔵庫で2〜8℃で15分間インキュベートします。 </li><li>洗浄するために10 7個の細胞あたりのPBS 1〜2 mlを加え、遠心10分間、300×gで。 </li><li>吸引し上清を完全にし、PBS500μlの10 8細胞まで再懸濁します。 </li></ol>白血球亜集団の3磁気選択注意：磁気ビーズセパレータと列の様々なメーカーの数から細胞表面マーカーによって白血球の特定の集団を単離するために利用可能です。分離は、1つまたは少数の正のマーカーにするか、不要な集団の枯渇後にネガティブ選択により基づく正の選択によって行うことができます。 （ すなわち、単球のためのCD4 + Tヘルパーリンパ球のために、またはCD14）は、1つのマーカーを使用して、正の選択を実行する一般的なアプローチは、以下に概説します。 <ol><li>準備し、製造元の指示に従って分離カラムを含むプライム磁気分離器。 </li><li>ラベルされたPBMCのサンプルを含むチューブを適用します。積極標識し、ラベル付け解除の収集のための2つの15ml遠心チューブを提供製造業者の指示に従って、細胞画分を編</li><li> 10 7細胞あたり1mlの培地を用いて、細胞数を15カウントおよび白血球完全培地中で正に選択白血球（ すなわち、CD4 + Tリンパ球またはCD14 +細胞）を再懸濁します。 </li></ol>増殖の評価のための白血球の4 CFSE染色注：CFSEは広くインビボおよびインビトロ 試験の両方のために、免疫学的研究において使用されています。私たちのプロトコルは、MSC / PBMC（または他の白血球）の相互作用のin vitroでの研究のために最適化されています。 CFSE in vitroでラベリングを行うに関わる一般的な手順は似ていますが、プロトコル14の具体的な用量およびタイミングの違いがあるかもしれません。これは、おそらくCFSE蛍光シグナルの強度に影響を与える可能性が使用される、多くの要因、 すなわち、使用される特定の細胞タイプ、細胞数とすることができます。 <ol&gt; <li> 10μM（CFSE-作業溶液）の所望の使用濃度になるようにPBSにCFSEストック溶液（10 mm）を希釈します。 CFSE-ワーキング溶液で標識するために10 7 細胞 （すなわち、PBMCを、またはT細胞）を準備します。 </li><li> 10分間、300×gで遠心分離し、細胞ペレットを得、上清を吸引します。 </li><li>穏やかに予め温めた（37°C）CFSE加工液1mlで細胞を再懸濁し、37℃で10分間細胞をインキュベートします。 </li><li>過剰CFSEを洗浄するために、予め冷却（4℃）RPMI培地を10％FBSを含有する（体積で）10倍で細胞懸濁液を希釈します。 5分間300×gの遠心分離により細胞を沈殿物と上清を捨てます。二回以上このようにして細胞ペレットを洗浄します。 </li><li>遠心分離によって細胞を再ペレット化し、細胞数15を数えます 。 1ミリリットル新鮮予め温め白血球完全培地中10 7細胞（PBMCまたはT細胞のいずれか）を再懸濁します。 </li></ol> Mの5共培養白血球や白血球の活性化とのSC <ol><li>せいぜい30分間37℃でプレ暖かいMSC完全培地（10％FBS（最適なMSCの成長のためにテスト済み）、1％L-グルタミン、およびDMEM低グルコース培地中で1％のペニシリン/ストレプトマイシン）。 </li><li> 24ウェルプレート中のMSCの完全培地1ml中の50,000個の細胞（MSC密度：25,000細胞/ cm 2）での種子のMSC 37℃のインキュベーター内の添付ファイルのO / Nのため、幹細胞を可能にするには、80％コンフルエンスに到達します。 </li><li>シードされたMSCの番号に基づいて、培地を吸引し、、CFSE標識したPBMCを追加（以前に標識してください上のプロトコル手順4を参照）で白血球完全培地1ml中（24ウェルプレートに、前日に播種）培養したMSCへ午前1時10分（細胞比）のPBMCに対するMSCの共培養比率。 </li><li>ウェルあたり1 mlの白血球完全培地の全容量中10μg/ mlの最終濃度まで、マイトジェンのフィトヘマグルチニン（PHA）、非特異的な白血球の活性化剤を加えます。 <ol><li> Alternativエリーは、特にTリンパ球の活性化のために刺激するために、α-CD3 / 28マイクロビーズを使用して、1のビーズ対Tリンパ球の比率を得るために、2つの細胞の共培養に追加する：1。 </li></ol></li><li>ネガティブコントロールのために、プレート50万のPBMC /ウェル（または特定のエフェクター白血球の人口;細胞密度25万白血球/ cm 2）でのみ1ミリリットル白血球完全培地を含む24ウェルプレート中、陽性対照のために、加えて、よくのPBMC /同じ数のメッキを10μg/ mlの最終濃度にPHAを加えます。 </li><li>第3回と共培養実験の5日目に、フルオレセインに適した488 nmの励起および発光フィルターを備えたフローサイトメトリー分析によって17（丸底チューブに入れ）CFSE標識白血球の増殖を評価。 注：フローサイトメトリー分析のために細胞内サイトカイン染色はまた、Mo等の白血球のサイトカイン発現プロフィールの変化を評価するために、この時点でこれらのCFSE標識白血球に行うことができますMSCのことでdulated。 CFSEは、フルオレセインのフィルタ適切に評価されているので、様々なサイトカインを評価するために選択された抗体は、フルオレセインまたは類似のスペクトル（クロロフィルタンパク質複合体（PerCPを）ペリジニンすなわち、フィコエリトリン）以外の蛍光色素にコンジュゲートされる必要があります。 </li></ol> 6.バリエーション：エフェクター抑制アッセイ磁気ビーズ選択、MSCによって誘発される免疫調節白血球活性化CFSE標識エフェクターCD4 + T細胞上の<ol><li> 6ウェルプレート中のMSCの完全培地3ml中250,000細胞（MSC密度：25,000細胞/ cm 2）での種子のMSC 37℃のインキュベーター内の添付ファイルのO / Nのため、幹細胞を可能にするには、80％コンフルエンスに到達します。少なくとも3つの6ウェルプレートは、亜集団の選択のための十分なMSC-共培養したPBMCを確保するために必要です。 </li><li>培地を吸引し、2.5×10 6細胞/ウェル（細胞密度でステップ1の通りが、6ウェルプレートで分離されたPBMCを追加25万ルを白血球完全培地3mLでukocytes / cm 2）でした。 37℃のインキュベーター中で48〜72時間のための共培養。 </li><li>磁気ビーズ選択セクション2-3のとおり、MSCによって誘発される免疫調節白血球（ すなわち、CD14 +細胞 ）の特定の集団を。 注：フローサイトメトリー分析のために細胞内サイトカイン染色は、白血球のサイトカイン発現プロフィール、 すなわち、の発現の変化を評価するために、この時点で行うことができるインターロイキン10などのMSCによって変調されます。 </li><li>様々な比でビーズ選択MSC誘導性の白血球：白血球完全培地1ml中の24ウェルプレート内のセクション2-4に従って生成されたCFSE標識同種CD4 + T細胞（250,000細胞/ cm 2のT細胞の密度）が追加しますすなわち、1：10、1：5,1：2、及び1：1（セルのセルに）比。 </li><li> 1：CD4 + T細胞を刺激するために、1のビーズ対細胞比を得るために、α-CD3 / 28共役マイクロビーズを加えます。 </li><li>ネガティブコントロール、プレート5の場合24ウェルプレート中0万CD4 + T細胞/ウェル（T細胞密度250,000細胞/ cm 2）でのみ1 mlの白血球完全培地と。 1：ポジティブコントロールのために、CD4 + Tの同じ数をメッキに加えて細胞/ウェルを、1のビーズ対細胞比を得るために、α-CD3 / 28共役マイクロビーズを追加します。 </li><li>共培養の3 日目に、フルオレセインに適した488 nmの励起および発光フィルターを備えたフローサイトメトリー分析により、16（丸底チューブに入れ）CFSE標識CD4 + T細胞の増殖を評価します。 </li></ol>

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We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

ヒト間葉系幹細胞（MSC）は、いくつかのextramesodermal系統5と同様に、骨、軟骨、および脂肪組織1-4の近軸中胚葉系統に分化することができる体細胞前駆細胞です。まず成人の骨髄から分離され、これらの多前駆細胞は現在臨床応用9-12に非常に適して表示され、強力な免疫調節特性を有することが示され、予想外に、多くの組織6-8で発見されています。免疫調節作用に関与する詳細なメカニズムは、積極的に特定の疾患実体の効果的なアプリケーションのために検討されています。免疫調節を評価するための最も簡単な方法の一つは、エフェクター白血球増殖13の抑制のための評価によるものです。刺激または活性化されると、このようなTリンパ球や単球などのほとんどのエフェクター白血球がprodigiously増殖します。免疫調節機能を評価することができるときの抑制増殖が明らかです。 伝統的に、エフェクター白血球の増殖は、DNAへの[3 H]チミジンの取り込みを検出することにより評価しました。しかしながら、この方法は、放射線及び使用後の廃棄の問題、ならびに必要に応じて複雑な装置に起因する重大な欠点を有します。細胞増殖を評価するための非放射性アッセイがあるが、カルボキシフルオレセインスクシンイミジルエステル（CFSE）アッセイは、複数の細胞型を含む共培養実験に特に有用である特定の細胞集団の同定を可能にするような他の利点を有します。 CFSEは、フローサイトメトリー分析によって評価することができる蛍光細胞の色素です。細胞が分裂するように、この細胞の標識の強度が比例し減少します。これは、全体的な細胞増殖の決意を可能にするだけでなく、蛍光がDETに困難になる前に8分割までの細胞分裂の数を評価するためにできるだけでなく電気ショック療法背景信号に対して。また、蛍光CFSEの安定性は、細胞が何ヶ月14までの視覚化することができるように標識された細胞のin vivoでの追跡が可能になります。 このアッセイはまた、磁性ビーズの表面マーカーの選択を行うことにより、CD14 +単球 15を生成する （IL-10）エフェクター白血球またはMSCによって誘導される免疫白血球、インターロイキン10などの特定の集団の免疫調節機能の特定のタイプを評価するために変えることができます前または適切な共培養後に目的の細胞集団。我々のプロトコルは、エフェクター白血球上のMSCの免疫調節効果を評価する基本的なアッセイ（フロー・チャートを図1に示す）と同種CD4 +エフェクター Tリンパ球上のMSCによって誘導される白血球の免疫調節の評価のためのこの基本的なアッセイの変形（図示フローチャートを説明します図4で）。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

図1は、実験の全体的なスキーマを示し、 図2は、位相差倒立顕微鏡によって可視化のような種々の細胞培養条件の外観を示しています。 PBMCおよび白血球が小さな丸い非付着細胞であるのに対し、MSCは、線維芽細胞、紡錘状形態を有する接着性の細胞です。これらの二つの形態学的に異なる細胞型が明らかに共培養に見ることができます。アッセイの終了時に、PBMCを（またはleuckoty...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

ヒト間葉系幹細胞（MSC）の免疫調節特性は、臨床応用のため、ますます関連現れます。蛍光色素カルボキシフルオレセインスクシンイミジルエステル（CFSE）で前染色MSCおよび末梢血白血球の共培養系を用いて、我々は、エフェクター白血球の増殖および特定の亜集団におけるMSCの免疫調節のインビトロ評価を記載します。

ヒト間葉系幹細胞（MSC）の免疫調節特性の評価

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

12.1K ビュー.  National Health Research Institutes (NHRI). ヒト間葉系幹細胞（MSC）の免疫調節特性は、臨床応用のため、ますます関連現れます。蛍光色素カルボキシフルオレセインスクシンイミジルエステル（CFSE）で前染色MSCおよび末梢血白血球の共培養系を用いて、我々は、エフェクター白血球の増殖および特定の亜集団におけるMSCの免疫調節のインビトロ評価を記載します。 

ビデオ: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

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

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

フローサイトメトリーによって負傷した脊髄組織における免疫細胞の定量的評価：セルの精製方法のための新しい使用法

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

感染を防ぐための新しいアプローチとして、PRP：準備と<em&gt;インビトロ</emPRPの&gt;抗菌特性

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

の検出のための磁気スターラー方法<em&gt;旋毛虫</em筋肉試料中&gt;幼虫

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

ヒト NK 細胞活性の評価の流れのフローサイトメトリーを用いた細胞毒性の試金

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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 ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

末梢血およびリンパ組織から主な人間の T 細胞のシナプスのインタ フェースの評価

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

ヒト幹細胞由来腸腸単層におけるM細胞様細胞の誘導分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...