Name: a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes
Uploaded: 2014-12-06T15:00:00.000+00:00
Duration: 7 min 29 s
Description: Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

この作品は、科学研究費（FNRS）、レス·エイミスドゥ研究所Bordetおよび、FNRS-操作Télévie、ベルギーのプランがん、フォンランボー-Marteaux、フォンJC HeusonとフォンBarsyベルギー基金をfromtheの補助金によってサポートされていました。

注：すべての標本は、各患者から得た書面によるインフォームドコンセントを得て研究所ジュール·ボルデの医学倫理委員会によって承認されたプロトコルを使用して取得した。 組織ホモジネートの調製<ol><li>切除組織（手術室から切除悪性および正常組織）を解剖即時のピックアップのために訓練を受けた者が病理学研究室である。腫瘍は、NANT（できるだけ腫瘍から最も遠い距離を撮影したもの）と正常組織断片を日常的に1内で処理される - ヒト組織のための標準的なバイオセーフティ手順を使用してBSL2実験室で外科的切除の3時間。プロトコルのフローチャートを図1に示されている。 </li><li>すべての組織片（通常NANTおよび腫瘍）を秤量し、長さ、幅、および高さ（長さ×幅×高さ）を測定する。これは、細胞集団のその後の正規化のための重要なステップは、抽出されたRNA 等である NOTE：可能な場合はサンプルサイズの範囲は、無脂肪と100〜10,000 mm 3である。 </li><li>組織が実際に腫瘍の一部であることを確認するために、H＆E染色のためにスライドガラス上の腫瘍断片をインプリントする。 <ol><li>腫瘍断片上のガラス顕微鏡スライドを押すと数秒間指で穏やかな圧力を適用することによってこれを行います。 </li><li>水中での洗浄工程に続いて2分間イソプロパノールでスライドを修正。マイヤーのヘマトキシリン​​で30秒のための組織を対比。 </li><li>水の6浴にスライドを洗ってください。フロキシンBで15秒間、2％を染色する。 </li><li>水1浴でイソプロパノールと仕上げの4風呂の水、続いて1浴で洗ってください。 </li><li> 1分およびドレイン用のイソプロパノール中でインキュベートします。キシレンの2浴場でクリア。 </li><li>キシレンベースマウント培地にマウントします。腫瘍細胞充実度（ 図2）のために調べます。 注：主に、腫瘍細胞が刻印スライドに固執 - 間質、リンパ系または広告ipose細胞はほとんどの腫瘍細胞（ 図2）の間にスペースを残して、残っていない。 </li></ol></li><li>滅菌を使用して-化学的に規定された、無血清造血細胞培地1mlを含む小さな培養皿で組織片を配置小片（2mmの2〜1）にそれを室温で（以下、媒体と呼ぶ）とダイス外科用メス。 </li><li>機械的解離のCチューブにすべてのもの（組織片+媒体）を転送します。 </li><li>パスツールピペットを用いて培地2mlでペトリ皿とメスをすすぎ、Cチューブ（解離のための媒体の体積= 3ミリリットル）にこれを追加します。 </li><li>単一細胞懸濁液に組織片を均質化するために、C管の機械的解離プログラムA.01（最も穏やかなプログラム）を使用します。装置内でCチューブを配置し、2回続けてプログラムを実行する（1サイクル= 25秒）。 注：この均質化手順は、他のTUM、ヒト乳房組織のために設立され、検証されているまたはまたは組織タイプは、別のプログラムを使用する必要があるかもしれませんし、最初にテストする必要があります。 </li><li>装置からCチューブを外し、50ミリリットルチューブに座った40μmのセルストレーナーに直接ホモジネートをデカント。 工程1.6と同様のパスツールピペットを用いて、セルストレーナーにC管に残っている液体を移す。 </li><li> 1ミリリットルマイクロピペットチップを用いて15ミリリットルチューブに濾過された液体を転送します。一時的にセルストレーナーとその50ミリリットルチューブを保つ。 </li><li>メディアの追加の3ミリリットルでCチューブをすすぎ、再びまだ50ミリリットルチューブに座ったセルストレーナーに、 ステップ1.6と同様にパスツールピペットを用いて、これを転送します。優しくクリーンなパスツールピペットまたはその後溶出液を汚染を避けるために捨てられる1ミリリットルチップでストレーナーのまわりでそれを移動して50ミリリットルチューブにunhom​​ogenized組織に閉じ込められた残液の最大量を絞る。 </li><li>逆さT上のセルストレーナーを配置彼元のC管とunhom​​ogenized組織がバックC管に降下するように、培地の3ミリリットルで洗い流し。 </li><li> A.01プログラムの2つのサイクルのためのステップ1.7のように再均質化する。 </li><li>再び最大圧搾50mlのチューブに着座細胞ストレーナーを通してこの第ホモジネートを入れ（ ステップ1.10のように）培地3mlで再びCチューブをすすぎ、50mlチューブに着座セルストレーナーにパスツールピペットで移すセルストレーナー中に閉じ込め残留結合組織から液体の量。 </li><li>この時点で、〜2.5ミリリットルの容積は、15mlチューブ中で、約50 mlチューブ9 mlである。 </li></ol>組織上清と細胞の2の分離<ol><li>室温で600×gで15分間15ml中ホモジネートを50 mlチューブを遠心する。 </li><li>きれいなチューブに15ミリリットルチューブからSNをデカントし、一時的に4℃で保存する。この上清=原発腫瘍、NANTまたは通常のTIssue SN（最終容量2.5ml）を、続いて清澄化し、将来の分析（下記参照）のために-80℃で保存する前に分注する。 </li><li> 50ミリリットルチューブから上清を捨てる。 </li><li>静かに1ミリリットル中の最終容量の両方で細胞ペレットを再懸濁します。簡単に言えば、最初の優しく（ハード面上にチューブをタップして）両方の管に細胞ペレットを破る。培地500μlで50ミリリットルで緩い細胞ペレットを再懸濁し、第二のペレットを再懸濁し15ミリリットルチューブにこの細胞懸濁液を転送します。セルの最大回復のための媒体の第2の500μlの一回、この手順を繰り返します。 </li><li>小さなチューブに移すの細胞懸濁液10μlを、トリパンブルー10μlのミックス（希釈1：1）と血球計数器を用いて生細胞の数を数える。 注：この時点で、細胞懸濁液の一部は、細胞サイズを評価するためにフローサイトメトリーによって分析することができ、粒度よりprecのための亜集団のマーカーの数が限られて、所望の場合事前の広範な分析や実験にホモジネートにおける相対的な細胞分布の伊勢評価。流れによる全ての分析は、フローサイトメトリー亜集団の正規化のためにCD45のラベルを組み込む。 </li><li>室温で10分間、300×gでの遠心分離によって細胞をペレット。腫瘍由来の細胞、NANT、または正常組織は今、さらに精製または分析のために準備ができている。手術と同じ日に行われたときにこれらの追加のステップは最高です。 注：フローサイトメトリー分析のために、すぐ1秒間ボルテックスし、細胞ペレットを赤血球溶解緩衝液0.4mlを添加し、室温で10分間の最小値をインキュベートすることにより抗体標識後に溶解されるべき残留赤血球を選別細胞ではない分析前の温度（光から保護）。 </li></ol>組織上清の3明確化<ol><li>遠心機で4℃で15分間15000×gで組織SN 1.5 mlチューブに。 </li><li>慎重にTを削除彼が触れたり、ペレットを乱すことなく、上清。希望する分量の数やボリュームに応じて、清潔なチューブ（またはチューブ）に移す。 </li><li>将来の使用のために-80℃で上清を保管してください。 </li></ol> 4.患者の血液<ol><li>手術前に、ヘパリン化チューブに静脈穿刺による制御の日として各患者から血液サンプルを収集する。血漿と軟膜を得るために、ブレーキをオフにして20ºCで10分間400×gで血液を遠心。 </li><li>血漿を除去し、20ºCで15分間万×gで遠心分離することにより、それを明確にする。将来の使用のために-80℃でアリコートとストアプラズマ。培地中のバフィーコートを希釈</li><li>前の即時分析を標準フィコール - ハイパック勾配遠心分離を用いて単核細胞を分離し、亜集団の単離、DNA / RNA /タンパク質抽出または凍結保存。 </li></ol> 5.フローサイトメトリー<ol><li>ラベル細胞の製造に応じて光から保護して4℃でのr命令。細胞ペレットを細胞溶解緩衝液0.4 mlで添加することによって抗体標識後の組織片由来の細胞懸濁液中に溶解赤血球。 </li><li>渦はすぐに1秒間、光から保護し、室温で10分間の最小値を、インキュベートする。洗浄することなく、データ取得のためのフローサイトメーターで標識細胞を渡します。 </li></ol>

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The authors declare that no conflict of interest exists.

この研究は、その後の細胞選別、抽出、凍結保存および/ ​​またはCD45 +集団の表現型分析のために酵素消化せず、正常および悪性の乳房組織ホモジネートの迅速な調製のために最適化された方法を記載している。この実験アプローチの目的は、密接にそれらのインビボ状態を反映し、手術室からの新鮮な組織の最小限の操作で、正常組織と比較しますTILの画像を生成すること...

The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.

<table><tbody><tr><td>GentleMacs Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>BD Medimachine is somewhat equivalent</td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td></td><td>or other standard table top centrifuge</td></tr><tr><td>Centrifuge 5417 R</td><td>Eppendorf</td><td></td><td>or other standard microcentrifuge</td></tr><tr><td>Esco Class II A2 Biosafety Cabinet</td><td>ESCO global</td><td></td><td>or other standard BSL2 hood</td></tr><tr><td>Inverted Microscope</td><td>Nikon eclipse TS100</td><td></td><td>or other microscope compatible for a hemacytometer</td></tr><tr><td>B&amp;uuml;rker Chamber</td><td>Marienfield&amp;nbsp;</td><td>640210</td><td>or other standard hemacytometer</td></tr><tr><td>Navios Flow Cytometer</td><td>Beckman Coulter</td><td></td><td>or other flow cytometer (8-10 color recommended)</td></tr><tr><td>GentleMacs C-Tube</td><td>Miltenyi Biotec</td><td>130-096-344</td><td>BD Medimachine uses Filcon</td></tr><tr><td>Cell Culture Dish</td><td>Sarstedt</td><td>72,710</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Disposable Scalpel</td><td>Swann-Morton</td><td>0510</td><td>or standard single use sterile scalpel</td></tr><tr><td>BD Cell Strainer 40 &amp;micro;m</td><td>Becton Dickinson</td><td>734-0002</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD Falcon Tube 50 ml</td><td>Becton Dickinson</td><td>352070</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD Falcon Tube 15 ml</td><td>Becton Dickinson</td><td>352097</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>BD FACS Tube 5 ml</td><td>Becton Dickinson</td><td>352008</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Sterile Pasteur Pipette 5 ml</td><td>VWR</td><td>612-1685</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>Microfuge Tube 1.5 ml</td><td>Eppendorf</td><td>7805-00</td><td>or other non-pyrogenic plasticware</td></tr><tr><td>X-Vivo 20</td><td>Lonza</td><td>BE04-448Q</td><td>serum-free medium recommended</td></tr><tr><td>Phosphate buffered saline</td><td>Lonza</td><td>BE17-516F</td><td>standard physiological PBS</td></tr><tr><td>Trypan blue</td><td>VWR</td><td>17942E</td><td>or other vital stain</td></tr><tr><td>VersaLyse</td><td>Beckman Coulter</td><td>A09777</td><td>for flow cytometry experiments</td></tr><tr><td>Fixable viability Dye eFluor 780</td><td>eBioscience</td><td>65-0865-14</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD3 FITC</td><td>BD Biosciences</td><td>345763</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD3 Vio Blue</td><td>Miltenyi Biotec</td><td>130-094-363</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD4 PE</td><td>BD Biosciences</td><td>345769</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD4 APC</td><td>Miltenyi Biotec</td><td>130-091-232</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD8 ECD</td><td>Beckman Coulter</td><td>737659</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD8 PerCP</td><td>BD Biosciences</td><td>345774</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD19 APC-Vio770</td><td>Miltenyi Biotec</td><td>130-096-643</td><td>for flow cytometry experiments</td></tr><tr><td>anti-CD45 VioGreen</td><td>Miltenyi Biotec</td><td>130-096-906</td><td>for flow cytometry experiments</td></tr></tbody></table>

a simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes

両方の自然免疫と適応免疫応答から腫瘍回避することを特徴とする免疫系を回避するために、悪性細胞の能力は、現在、癌の重要な特質として受け入れられている。乳癌に関する我々の研究は、腫瘍浸潤リンパ球は、腫瘍の進行および患者の転帰に果たす積極的な役割に焦点を当てています。この目標に向けて、我々は彼らの本来の状態にそれらが近接し、評価するための努力で正常と異常な組織から無傷のリンパ細胞の迅速な単離のための方法論を開発しました。酵素で消化された組織と比較して表面の受容体の発現を維持しながら、ホモジネートを、機械的解離のショーを使用して増加した生存率および細胞の回復の両方を用意しました。さらに、残りの不溶性物質の酵素消化は、プライマリホモジネートにおける定量的および定性的測定が可能性が高い純粋に組織fragmで浸潤亜集団を反映していることを示す追加のCD45 +細胞を回復しませんでしたENT。これらのホモジネート中のリンパ球様細胞は容易に免疫学（表現型は、増殖など ）または分子（DNA、RNAおよび/ ​​またはタンパク質）に近づく使用して特徴付けることができる。 CD45 +細胞は、亜集団の精製、in vitro増殖または凍結保存のために使用することができる。このアプローチのさらなる利点は、ホモジネートから一次組織の上清を、サイトカイン、ケモカイン、免疫グロブリンおよび正常および悪性組織中に存在する抗原を特徴付け、比較するために使用することができることである。このプロトコルは、ヒト乳房組織に極めてよく機能し、正常と異常組織の多種多様に適用可能であるべきである。

市販の組織解離溶液またはコラゲナーゼの様々な実験用混合物は、DNaseおよび/またはヒアルロニダーゼ阻害剤のいずれかと組織断片の酵素消化は、細胞の表面上の受容体の多種多様を切断する。最初に乳房腫瘍浸潤CD4 + T細胞に焦点を当てた我々の研究は、急速による標準的な酵素消化プロトコル4-8を用いた表面CD4受容体の切断に大きな技術的な問題を提示した。我々は、高?...

Watch this Scientific Journal Video about A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes at JoVE.com

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

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 ...

a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human

Research

JoVE Journal

Immunology and Infection

19.9K ビュー.  Université Libre de Bruxelles. 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.

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

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

免疫・感染学

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

共培養からの侵襲的細胞亜集団のリアルタイム検出と捕捉

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

がん研究

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

医学

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

解剖されたインプラント組織の免疫機能解析のための高次元フローサイトメトリー

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工学

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue

Source: Garaud et. al., <a href="https://www.jove.com/v/52392/a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human">A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes.</a> J. Vis. Exp. (2014).
This video describes the non-enzymatic dissociation of fresh human tissue to obtain viable cells. The technique is used for qualitative and quantitative analysis of CD45 positive cells (lymphocytes/leukocytes) present in various normal and malignant human tissues.

機械的解離:組織から生細胞を得る方法

Source: Garaud et. al., A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes. J. ...

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

要約

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この動画の章

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

共培養からの侵襲的細胞亜集団のリアルタイム検出と捕捉

ネズミリンパ節間質細胞の単離

表現型分析のためのヒト皮膚からリンパ球単離と

母体·胎児界面におけるマウス組織からの白血球の分離

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

解剖されたインプラント組織の免疫機能解析のための高次元フローサイトメトリー

Mouse Wound Models and Preparation of Single-Cell Suspensions

機械的解離:組織から生細胞を得る方法