研究は、トランスレーショナルリサーチのウォレス時間コールター財団早いキャリア賞によってサポートされています。血液サンプルの収集とヘルプのためのティムアンドリュース、小児血液/腫瘍学、のおかげ。また博士はサムヴェルハウゼン、フローサイトメトリーコア、フローサイトメトリーのサンプルを実行するとヘルプのためのジェームズグラハムブラウンがんセンター、おかげで。

パート1。マイクロ流体デバイスの製作マイクロ流体の溶解装置のシリコンのマスターの金型はルイビルクリーンルームの大学での機器で標準的なフォトリソグラフィ技術を使用して作成された[1]。 AutoCADは（オートデスク株式会社、サンラファエル、カリフォルニア州）チャネルの負のレプリカを作成する（輝いているイメージング、コロラドスプリングズ、コロラド州）透明度のマスクを生成するために使用されていました。マイクロ流体の溶解装置は、エラストマー、ポリ（ジメチルシロキサン）（ダウコーニング、ミッドランド、MI PDMS）とソフトリソグラフィ技術を用いてシリコンマスター金型から製造された。複製されたチャネルを持つエラストマーがリリースされ、チャネルのアクセスホールは、20ゲージの針（スモールパーツ、ミラマー、フロリダ州。）でパンチされた。 PDMSのウェハは、不可逆的に酸素プラズマを介してスライドガラスに接着し、実験のためにテストされています。 パート2。溶解装置を実行するためのプロトコルマイクロ流体デバイスを作製し、テストされた後、赤血球溶解の実験では、開始する準備が整います。次のセクションでは、マイクロ流体デバイスのためのプロトコル。 2.1サンプル集<ol><li> two 2ミリリットル緑トップvaccutainersの抗凝固剤としてヘパリンを持つ患者の前腕の静脈に、肘正中皮静脈から血液サンプルの4ミリリットルを収集する。 </li><li>それが外れ成熟した内皮細胞を（偽陽性）が含まれているとして、最初の2 mlチューブを捨てる。 </li><li>上下に回すことで血液とヘパリンを混合する第2のチューブを再懸濁し、氷上に即座に保存。 </li><li> 1.5mlのエッペンドルフチュー​​ブへの血液の1 mlを入れる。 （コレクションの一時間以内）に血液サンプルを処理します。 </li></ol> PBS 2.2総理マイクロ流体デバイス<ol><li>ガラスに結合マイクロ流体デバイスを取得します。入口と出口で少し大きめの直径（図1）の圧入アクセスチューブ（スモールパーツ、ミラマー、フロリダ州）。 </li><li>ミクロマクロをインターフェイスに提供し、入口の各チューブに30ゲージの注射針を（小さな部品、ミラマー、フロリダ州）に取り付けます。 </li><li> 1X PBSで1 mlのシリンジを記入し、シリンジの針をフリックで気泡を取り除くために確認してください。マイクロ流体デバイスの入口1にPBS -ロードされた1 mlのシリンジを接続してください。解決策は、入口2から流れ出すまで、手で軽く1X PBSを含む注射器を押し込み、すぐに標準的なオフィスバインダークリップで口2をクランプ</li><li>流体がマイクロ流体デバイスの出口ポートに到達するまで、1X PBSを押し続けます。解決策は、コンセントから流れ出すと、別のバインダークリップでアウトレットチューブをクランプする。 </li><li> 1X PBSが口3から流出するまで、1 mlのシリンジを押し続けると、別のOfficeバインダークリップで口3ポートをクランプする。マイクロ流体デバイスは、今や完全に準備されている。 </li></ol><img src="/files/ftp_upload/1656/1656fig1.jpg" alt="図1" /> 図1の各ソリューションとコンセントのための入口を示す装置の概略図。 シリンジポンプとソリューション2.3準備<ol><li>ポンプのキャリブレーションを行うには、次の手順を実行します。 <ol class="lalpha"><li>大規模なハーバードのシリンジポンプ（ハーバード大学、PHD 2000分の22の部品番号702000）の場合：22.5ミリメートル、流量600μL/ minのためにDiameterコンフィグレーションを設定します。 </li><li>小さなハーバードのシリンジポンプ（ハーバード大学、ピコプラス部品番号702213）の場合：4.61ミリメートル、流速20μL/ minにDiameterコンフィグレーションを設定します。 </li></ol></li><li>滅菌脱イオン水を使用すると、ワン30mlのシリンジを埋める。これは、50 mlコニカルチューブから直接解決策を引き出すことによって達成することができます。 （注射器にラベルを付ける） </li><li>手順9で説明した手順を使用して、2Xリン酸緩衝-生理食塩水（PBS）、2％パラホルムアルデヒド（PFA）で2 30mlのシリンジを埋める。 （注射器にラベルを付ける） </li><li>手順9で説明した手順を使用して、15 mlコニカルチューブに格納されたアリコートから1X PBSで1 mlの注射器を埋める。 </li><li>入口2と入口3〜30ミリリットル2X PBSのシリンジに30 mLの脱イオン水のシリンジを接続し（チューブまたはマイクロ流体デバイスで使用するドンtのトラップの気泡を確認してください）​​。入口2と3とアウトレットチューブからクランプを外します。入口1に1X PBSを1mlのシリンジを接続してください。 、液体を注射器の針を充填シリンジを接続し、気泡を除去するために注射針をフリックで気泡を導入することは避けてください。 </li></ol> 2.4赤血球溶解<ol><li>以下に概説されるよう適切なハーバードポンプのシリンジのすべてを設定します。 <ol class="lalpha"><li>大規模なハーバードのシリンジポンプに一緒に2つの30 mLシリンジ（1つ滅菌、脱イオン水と2X PBS、2％PFAで他を含む）を設定します。 </li><li>小さなハーバードのシリンジポンプに1 mlの注射器を（1X PBSを含む）を設定します。 </li><li>廃棄物のコレクターでアウトレットチューブを置きます（Wラベル付けされている50 mlコニカルチューブASTE）。 </li></ol></li><li>大規模なハーバードのシリンジポンプ（30 mLシリンジ付き）をオンにして、それを1分間実行してみましょう。 （は泡とデバイスまたはチューブのない漏れがないことを確認してください）​​。 </li><li>小さなハーバードのシリンジポンプの電源を入れ、追加分のために実行してみましょう。 （は泡とデバイスまたはチューブのない漏れがないことを確認してください）​​。ポンプを停止します。マイクロ流体デバイスは使用可能な状態になります。 </li><li>小さなハーバードのシリンジポンプから1X PBSを含む1 mlのシリンジを取り外します。 </li><li>血液サンプルを取得した後、気泡をトラップすることなく、滅菌1X PBSの0.05 mlを1 mlの注射器を埋める。次に、エッペンドルフチュー​​ブから得られた血液0.5mlをシリンジを埋める。 （1 mlの注射器は血液と1 × PBS緩衝液の0.55 mlの最終容量が含まれます。） 注 ：血液、0.05 mlのPBSの混合を避けるために充填しながら注射器を垂直に保つ。 </li><li>入口1に血液を含有する注射器を接続し、小さなハーバードのシリンジポンプ（シリンジをポンプに垂直マウントされている）上（装置にチューブを介して血液を押し避ける）慎重にシリンジをマウントします。 </li><li>廃液チューブからアウトレットチューブを取り外し、その場所に新しい50 ml遠心チューブに入れ、氷のバケツの上に置いてください。 </li><li>最初の大ハーバードのシリンジポンプのスイッチを入れ、それが1分間実行してみましょう。 50 ml遠心チューブにコンセントからのサンプルの収集を開始します。 </li><li>小さなハーバードシリンジポンプのスイッチを入れる。 </li><li> 1mlの注射器で血液サンプルが完全にデバイスを通過するとしたら、両方のポンプを停止する。これは約20分かかります。 </li><li>ブレーキオフ、室温で350 xgで5分間採取した試料を遠心分離します。 </li><li>白色ペレットの反対側にピペットチップを配置することによって、上清を取り除きます。ホワイトペレットを乱すことなく、可能な限り、特に赤血球の破片上清をできるだけ取り除く。 </li><li>上下にピペッティングして流れの緩衝液1ml（1X PBS、1％BSA [ウシ血清アルブミン]、2％PFA）でサンプルを再懸濁します。 1.5mlのエッペンドルフチュー​​ブにサンプルを移す。 </li><li>室温で4 × gで5分間遠採取されたサンプルを。上清を除去する前と同じ精度を使用してください。 </li><li>ボルテックスで流れの緩衝液1mlでサンプルを再懸濁します。 </li></ol>フローサイトメトリー分析用2.5準備<ol><li>フローサイトメトリーを用いたサンプルの分析のために、フローサイトメトリーチューブにサンプルの100μlを加える。 </li><li>試料100μlに指定された抗体を（正しい濃度を確保する）を追加。 </li><li>サンプルは40℃で30分間インキュベートすることができます</li><li>フローサイトメトリーの前に流れの緩衝液で2回洗浄する。洗浄工程は、フローバッファを250μlを加えるボルテックス、350 xgで5分間遠心分離し、フローサイトメトリーチューブの一つの流体の回転と上清除去を含みます。 </li><li>フローバッファ250μlのでペレットを再懸濁します。サンプルは、フローサイトメトリーによって分析の準備ができています。 </li></ol>代表的な結果全血は、赤血球と豊か循環有核細胞（のCNC）からなくし、マイクロ流体の溶解装置と後処理のプロトコルを介して実行されています。理想的な結果は、独立したCNCの人口を持つクリーンなフローサイトメトリーの散布図が得られます。図2と図3の下に表示さ溶血することなく、前方散乱光（FSC）対側方散乱（SSC）の光のような軸を持つマイクロ流体プロトコルによる溶血による全血サンプルの散布図の比較です。それは、マイクロ流体濃縮プロトコルがクリーンフローサイトメトリーの散布図を得るために必要であることが分かる。 <img src="/files/ftp_upload/1656/1656fig2.jpg" alt="図2" /> 図2。FSCとSSCの軸を持つ赤血球溶解せずに全血サンプルのフローサイトメトリーの散布図。 <img src="/files/ftp_upload/1656/1656fig3.jpg" alt="図3" /> 図3。FSCとSSCの軸を持つ集団で標識されたマイクロ流体溶血と核細胞を循環のフローサイトメトリーの散布図。リージョン1（赤）は顆粒球を表すリージョン3（青）、領域2（緑）が単球を表し、リンパ球を表しており、リージョン4（紫）が特徴とされていない。 ときに必要なプロトコルのピペットによる赤血球の破片の除去も重要である。下の図4には、赤血球の破片の余剰を示す散布図である。図2のような散布図を曇らせるではありませんが、一つにはデータ分析の赤血球破片のために追加されたイベントを考慮する必要があります。 <img src="/files/ftp_upload/1656/1656fig4.jpg" alt="図4" /> クラスタとして見られる赤血球の破片を描いた図4。フローサイトメトリー散布図、地域5（シアン）のイベントの。 特定の抗体の表現型マーカーの染色細胞はまた、特徴的な結果を生成します。リンパ球、単球、顆粒球：以下の図では3つの異なる白血球集団のための抗体の表現型の汚れです。図5は、軸CD3とCD4にプロットされたリンパ球集団を表しています。単球および顆粒球は、それぞれ、FSCに対するCD14とCD66bのような軸を持つ6〜7図に示されています。 <img src="/files/ftp_upload/1656/1656fig5.jpg" alt="図5" /> 図5。フローサイトメトリーの散布図は、CD3とCD4の軸を持つリンパ球を描いた。 <img src="/files/ftp_upload/1656/1656fig6.jpg" alt="図6" /> 図6。フローサイトメトリーの散布図は、FSCとCD14の軸と単球を描いた。 <img src="/files/ftp_upload/1656/1656fig7.jpg" alt="図7" /> 図7。フローサイトメトリーの散布図は、FSCとCD66b軸を持つ顆粒球を描いた。

<ol>
	<li>White, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+microfluidic+leukocyte+enrichment+protocol+in+mild+phenotype+sickle+cell+disease+(SCD.">Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD.</a> Biomedical Microdevices. 11 (2), 477-483 (2009).</li><li>Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Microfluidic+Device+for+Rapid+Erythrocyte+Lysis.">Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis.</a> Anal. Chem. 76 (21), 6247-6253 (2004).</li></ol>

自動化されたマイクロ流体血液溶解プロトコルが報告されている。プロトコルの中で注目に値する重要な手順は次のとおりです。セクションP.2.1で、それはサンプルが外れ成熟した内皮細胞が含まれている偽陽性として機能するように収集された最初の血液バイアルを廃棄することが重要です。また、コレクションの1時間以内に血液サンプルを実行することを確認してください。プライミン?...

automated microfluidic blood lysis protocol for enrichment of circulating nucleated cells

この報告書では自動化されたマイクロ流体血液溶解装置用プロトコルが詳しく説明されています。白血球と内皮細胞を含む有核細胞（のCNC）、循環、個々の免疫状態に関する更新状況のための理想的なプラットフォームを提供します。マイクロ流体のプロトコルは、選択的な細胞の損失と、ユーザーが介在するステップによる試料調製の変動なしでCNCの濃縮が可能になります。簡単に言えば、プロトコルは、デバイス作製、サンプルの収集、デバイスのセットアップ、およびマイクロ流体チャンバーを介して血液を実行しているが含まれています。デバイス内で全血は急速に50ミクロン× 150ミクロンマイクロ流体チャネル内の約10秒間、脱イオン水と混合される。この時点ではスパンの赤血球は低張条件により可溶化されています。チャネルの底部のヘリンボーン構造が完全に混合し、一定の環境への細胞の露出を確認してください。残りの細胞は、装置の出口で等張条件に戻さ2％のパラホルムアルデヒドを用いて固定し、CNCのから赤血球残骸を分離する遠心分離し、フローサイトメトリーによる染色と分析のために流れの緩衝液に懸濁している。結果は保存されているCNC人口クリーンフローサイトメトリーの散布図を示す。このデバイスとプロトコルの重要性は、CNCの個体群の解析による疾患の病因の研究と理解に来る。したがって、自動化、有効性、およびマイクロ流体プロトコルのシンプルさが実証されています。

Watch this Scientific Journal Video about Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells at JoVE.com

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

自動化されたマイクロ流体デバイスは、セルの損失なしで高品質のサンプルの分離を確実に溶血を経由して末梢血からの有核細胞の濃縮を循環させるために開発されました。

有核細胞を循環の濃縮のための自動化されたマイクロ流体血液溶解プロトコル

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

automated-microfluidic-blood-lysis-protocol-for-enrichment

Research

JoVE Journal

Biology

12.3K ビュー.  University of Louisville. 自動化されたマイクロ流体デバイスは、セルの損失なしで高品質のサンプルの分離を確実に溶血を経由して末梢血からの有核細胞の濃縮を循環させるために開発されました。

ビデオ: Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell Technologies社からRosetteSepキットと人間の血液からNK細胞の濃縮

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

生物学

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫蛍光による非小細胞肺癌患者からの循環腫瘍細胞の半自動PD-L1特性図と列挙

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

がん研究

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

ヒト白血球と血小板の単離と評価のためのプロトコル：生体エネルギーと酸化バースト

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

免疫・感染学

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

サイトカインキャプチャシステムを用いた臨床応用のためのサイトメガロウイルス特異的T細胞の自動化された細胞濃縮

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

抗腫瘍特性を有する好中球の単離とキャラクタリゼーション

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

閉ループ慣性マイクロ流路を用いた患者由来気道分泌からラベル無料好中球の濃縮

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...