Name: isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens
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Description: Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

手術患者からのヒト組織を破棄し、初代培養物の確立はテキサス大学医学部、ニューメキシコ制度審査委員会の大学によって承認された取得するためのプロトコル。ヒト組織標本の調達のための一般的な要件について説明します。 MFsは凍結組織から単離することができるので、バイオバンクや商業ベンダーはまた、実行可能な選択肢であることに注意してください。 1.ヒト組織を入手<ol><li>承認のための制度審査委員会へのヒト結腸組織を得るためのプロトコルを提出してください。 </li><li>研究のためのヒト組織を得るために、一般的な外科医および/または結腸直腸の外科医との連携を確立します。彼らは診断のために必要とされず、研究のために指定することができる組織を決定するように、病理部門との連携も必須です。 </li><li>その後、新生物の組織を提供するために、病理学者に助言し、grosslY正常粘膜腫瘍から5cm以上。直ちに氷冷洗浄培地中のサンプルを配置し、氷上に置きます。病理学者は、研究のために安全に与えることができる組織の量を決定します。 MFの単離のために必要な最小限の組織は、1.5ミリメートル2です。 </li><li>将来の単離のために-80℃で一次培養または凍結と場所を確立するために、直ちに得られた組織を使用してください。 <ol><li> -80℃で凍結培地（下記参照）、および店舗1mlの極低温管に置き、2 mmの2個-後単離するために、凍結した場合、約1内に組織を切断します。注：このプロトコルを利用して、4年以上の凍結組織からのMFsの成功の分離が観察されています。 </li></ol></li></ol> 2.試薬を準備<ol><li>コラゲナーゼ溶液の場合： の Ca 2+をハンクス平衡塩溶液（HBSS）で100 U / mlのコラゲナーゼI、IIおよびIVを準備/ Mgが2+（材料表を参照してください </stronG&gt;）。注意：コラゲナーゼアクティブユニット濃度および純度は、組織消化および/または粘膜の細胞表面上のエピトープの保全の効率化を妨げる可能性がある、ベンダーからベンダーに変動することがあります。これらの潜在的な落とし穴を防ぐ純度の最も高い（≥95％）とコラゲナーゼを使用して、アクティブユニット濃度に基づいて、ストック溶液を調製しました。 </li><li> 10 mg / mlの（3.55 UA / mg）でで水ベースのDNaseストック溶液を準備します。 </li><li>ウォッシュメディアを準備します（MEM：89ミリリットルを、抗生物質 - 抗真菌、100X：1ミリリットル;熱不活性化FCS：10ミリリットル） </li><li> MEM：線維芽細胞増殖培地を準備する500ミリリットルのボトルを。 L-グルタミン（200 mM）の5 mlでMEM非必須アミノ酸、100×溶液5 mlでシプロフロキサシン塩酸（10 mg / mlで）：570μL;抗生物質 - 抗真菌、100倍液：10ミリリットル。ピルビン酸ナトリウム（100 mM）の5 mlで熱不活性化FCS：50ミリリットル</li><li>フィルタ-STERをその後、10％ジメチルスルホキシド（DMSO）を添加した線維芽細胞増殖培地（試薬2.4）：凍結培地を調製します0.22μmのフィルターでilize。 </li></ol> 3.酵素的にダイジェストヒト組織<ol><li>組織とメディアが解凍されるまで組織を温かい（〜37℃）の水の凍結メディア、場所クライオバイアル中で凍結された場合。 </li><li> 4の場合-組織の2ミリメートル2枚の5、のCa 2+ / Mg 2+をすることなく、HBSSで2回10mlの組織を洗います。 </li><li>組織は、重力によって解決したら、慎重にサンプルを回転させずに、上清を捨てます。 </li><li>サンプルへのコラゲナーゼ溶液10mlを加え、サンプル解離のための組み込みのローターを滅菌、DNアーゼ/​​ RNaseフリーのチューブに組織液の混合物を移します。上皮細胞の基底面を囲み、粘膜固有層の非細胞画分を形成する細胞外マトリックスを解離するためにコラゲナーゼを使用してください。 </li><li>しっかり近いチューブ（例えば、GentleMACs）解離のスリーブ上に逆さまに取り付けます。ラボは、トンへのアクセス権を持っていない場合彼は、同等の結果を得るために、各消化工程の時間を増加させる、装置を前述しました。 </li><li>プログラムh_tumor_01を実行し、予め設定されたプロファイルは、マシン（ - ランあたり268回と4,000 rpmの範囲の断続的なパルスで36秒の合計時間、1000〜）に含まれています。 </li><li>プログラムの終了後、解離からチューブを外します。 </li><li> 140 rpmで振とう機上で連続回転下37℃で45分間サンプルをインキュベートします。注：組織サンプルの量を変化させるため、消化の時間は比例的に減少または増加させることができる（ すなわち 、組織のより大きな量をさらなる時間を必要とするかもしれません）。 </li><li>解離のスリーブ上に逆さにチューブを取り付けます。 </li><li> （ - 1回あたり235回で、4,000 rpmで千に至るまで断続的なパルスで、37秒の合計時間）を選択して、プログラムのh_tumor_02を実行します。 </li><li>プログラムの終了後、解離からチューブを外します。 </li><li> DNアーゼストック溶液50μlを追加。 /解離死細胞および凝集をセルにつながる細胞破片のDNA画分を除去するためのDNAseを使用してください。 </li><li> 60 rpmで振とう機上で連続回転下、37℃で30分間サンプルをインキュベートします。 </li><li>再び解離のスリーブ上に逆さにチューブを取り付けます。 </li><li> （ - 1回あたり168回で、4,000 rpmで千に至るまで断続的なパルスで、37秒の合計時間）を選択して、プログラムのh_tumor_03を実行します。 組織が完全に消化されていない場合は、20℃で10分間、250×gで遠心分離後、上清を捨て、細胞解離溶液2ml中に再懸濁し、10分間室温でインキュベート。注意して​​ください。 </li><li>滅菌70μmのセルストレーナーを通して細胞懸濁液を渡します。 </li><li> 20℃で10分間、250×gで遠心細胞懸濁​​液。完全に上清を吸引。 </li><li>洗浄細胞のCa 2+ / Mg 2+をなしHBSSで2回25mlでペレット、上清を捨てます。 </li><li>最後に先立ちましたhは、実験室で利用可能な自動細胞計数システムを使用して、または手動で血球計数器を用いて細胞数を数えます。 MF初代培養生成するステップ3.19.2進行フローサイトメトリーを用いてのMFsの解析やソートに、ステップ3.19.1進行。 <ol><li>分離及び純粋なMF培養物の増殖（ 図1）のために、培地3mlに4×10 6個の細胞にまで播種するために必要な繊維芽細胞の分離媒体の適切な量で、最後の遠心分離、再懸濁細胞ペレットから上清を捨て、ウェルあたりで6ウェル、細胞培養処理プレート。 3.20に進みます。 </li><li>分析のために、または、フローサイトメトリー24で線維芽細胞の分離培地の2 ml中に2×10 6までの配置だけでなく、低結合プレートおよび5％CO 2、37℃でO / Nインキュベート使用してのMFsのソート。これは、酵素的手順によって影響され得る細胞表面エピトープを回復することです。その後、細胞懸濁液を回収し、回収した細胞をカウントし、続行免疫染色のための分析8フローサイトメトリー。 </li></ol></li><li> 5％CO 2、37℃で細胞を増殖させます。 〜80％コンフルエントのMF単層（ 図1D-E）が形成されるまで3日-ごとに2メディアを変更します。 </li><li>比1で6ウェルプレートからでT25フラスコ内の通路細胞：1と〜のための10の成長 - 100％の密集度 - 80を達成するために、線維芽細胞増殖培地で14日。 </li><li> 2：1の比率でT75細胞にT25から細胞を継代することにより文化を展開します。細胞がコンフルエントに達すると、先に説明したように5フローサイトメトリーで使用して孤立したMF純度の分析のための1フラスコを使用しています。 3：1の割合で、MF文化の別のT75フラスコを凍結または通過。 注意：EpCAM-、CD31-、CD45 - 、ビメンチン+、α-SMA +、CD90 +：フローサイトメトリーで分析すると、それは間葉起源のGI粘膜の間質MFは文化の中で成長すると、次の表現型を持つことになりますときにことが期待されます。 </li></ol>

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The authors have nothing to disclose.

このプロトコルは、唯一の研究用途のために開発されたが、潜在的な癌予後バイオマーカーおよび治療標的としての間質細胞の成長の重要な重要性に照らして、後で使用するために「機能的」生物試料を凍結する能力は重要な利点を提供しています。これは、個別化医療10、11の開発を進めるのに役立つなどの追加のツールとして機能し得る臨床バイオレポジトリーを作成するための?...

筋線維芽細胞/線維芽細胞（mfsが）胃腸（GI）粘膜内の細胞の豊富な集団です。これらの間質細胞だけ上皮の下に位置し、腸内の粘膜固有層内の相互接続ネットワークを形成しています。 mfsがばかりでなく、細胞外マトリックスの沈着の原因であるが、パラクリン調節を介して電解質輸送、回復、および隣接する上皮1,2のバリア機能に影響を与えることができる。さらに、mfsが炎症および組織において重要な役割を果たすことが示されています3、4改造。筋線維芽細胞は、それらがまた、癌関連線維芽細胞として知られている腫瘍の微小環境の重要な一部であり、腫瘍細胞増殖に寄与し、癌幹細胞ニッチ3として機能します。新興データがmfsも、地元の抗原提示細胞として機能し得ることを示唆しています。さらに、のMFS機能自然免疫と適応免疫応答の重要な調節因子として、のproduサイトカインおよび増殖因子5の様々なcing。 健康な個体では、のMFS細胞は、これらの細胞は、ビメンチンに対して陽性である。間葉系幹細胞から分化し、それらの細胞表面、間葉系マーカー、CD90 3,5に発現すると考えられているが、上皮細胞および造血細胞マーカー、のEpCAMおよびCD45について陰性、それぞれ。筋線維芽細胞は、線維芽細胞の活性化形態であることが示唆されており、α-SMA 5の発現により活性化されていない線維芽細胞から区別することができます。 過去10年間、人間の結腸粘膜から筋線維芽細胞を単離するための複数のアプローチが発表され、大部分はもともとMahida ら 5-8に記載された方法に基づいています。個々の研究は、様々な腸粘膜から単離手順を報告しているが、GI管の複数の領域からのMFsを単離するための普遍的なプロトコル（ すなわち 、胃、小およびlARGE腸粘膜）が公開されています。本明細書に提示プロトコルがテストされ、正常に上記の組織のすべての3つのタイプに使用されています。 。また、冷凍GI粘膜からのMFsを単離するための手順が報告されていません。 ここでは、酵素消化に基づいて最適化された方法を提示し、同時に培養のための人間の腸粘膜のMFsの分離を可能にし、新たに消化し、単一の細胞、粘膜製剤中のフローサイトメトリー分析。この技術は確実にMF表現型と初代培養物を生成します。また、同様の方法は、同様に胃および小腸組織の凍結標本からのMFsを単離することができます。新鮮GI組織から筋線維芽細胞の単離は、以前に記載されています。しかし、冷凍標本の利用は、多くの利点を提供します。すなわち、研究者はcapabiを持っている世界中の協力者の任意の数から収集して保存組織することができるだろう凍結組織サンプルを出荷するリティ。また、研究者は、現在の実験スケジュールと競合への単離のための組織を処理し、手術室から廃棄された組織の収集および/または内視鏡検査スイートと即座に見つけることができます。また、手術の予測不可能な性質のために、組織の調達が処理組織のために残された時間が制限されている、非常に遅めの時間に発生することがあります。後の処理のために組織を凍結することは、これらの課題を改善します。 最後に、これらの方法は、正常結腸、胃および結腸直腸癌および炎症性腸疾患のような疾患状態における小腸筋線維芽細胞の単離に利用されています。

<table><tbody><tr><td>MEM, 1x</td><td>Corning</td><td>MT-10-010-CV</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100x</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>Corning</td><td>61-277-RF</td><td></td></tr><tr><td>L-Glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DL-Dithiothreitol solution (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Cellgro</td><td>46-034-CI</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002139</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type I</td><td>Sigma-Aldrich</td><td>C1639</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type II</td><td>Sigma-Aldrich</td><td>C1764</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Accumax cell dissociation solution</td><td>Sigma-Aldrich</td><td>A7089</td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Cell Strainer (70 &amp;micro;m)</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>PE Mouse Anti-Human Vimentin</td><td>BD Biosciences</td><td>562337</td><td>Clone RV202</td></tr><tr><td>FITC Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma-Aldrich</td><td>F3777</td><td>Clone 1A4</td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Biosciences</td><td>559869</td><td>Clone 5E10</td></tr></tbody></table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

線維芽細胞/筋線維芽細胞（mfsが）病因におけるそれらの役割と腫瘍微小環境の両方の創傷治癒やプロモーションへの貢献のためにますます注目を集めています。現在、胃腸（GI）組織からのMFsを単離するための多くの技術がありますが、このプロトコルは、凍結組織からこれらの間質細胞の単離の新規要素を紹介します。 GI組織標本を凍結するだけでなく、世界中の共同研究者、バイオバンク、および商業ベンダーからサンプルを取得するために研究者が、それはまた、新鮮なサンプルの遅延処理を可能にすることができます。記載されているプロトコルは、一貫して、マーカーCD90、α-SMAおよびビメンチンを表現MF表現型と特徴紡錘状細胞が得られます。これらの細胞は患者のサンプルから誘導されるように、一次細胞の使用は、疾患状態、すなわち癌および炎症性腸疾患に密接に模倣するのMFsの利​​点を与えます。この技術は蜂を持っていますnは、胃、小腸、および結腸MF初代培養世代で検証。初代MF培養は、通路の数を超える実験の広大な配列で使用することができ、それらの純度は、両方の免疫細胞化学によって評価し、フローサイトメトリー分析。

この酵素消化プロトコルを使用して、我々は一貫して分離し、GI外科標本や生検（ 図1）からMF人口腸CD90 +粘膜の間質細胞を成長させることができました。 6ウェルプレート（ 図1A-B）中の単核細胞懸濁液を播種した後、5 -可視MFコロニー増殖は2日目に観察されました。 11（ 図1C-D） - 7日目までに70％コンフルエンス- MF初代?...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

人間の冷凍消化管標本からCD 90 +線維芽細胞/筋線維芽細胞の単離


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

isolation-cd-90-fibroblastmyofibroblasts-from-human-frozen

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.5K ビュー.  University of Texas Medical Branch. Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production. 

ビデオ: Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

発生生物学

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

FACSによるマウス皮膚線維芽細胞の分離

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

蛍光活性化細胞選別によるヒトの皮膚からの乳頭状および網状線維芽細胞の単離

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遺伝学

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

病気のモデリングのための筋生検からの患者由来の細胞株の単離と不死

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

医学

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工学

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

生物学

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超音波増強原発性成人線維芽細胞分離

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

マウスおよびヒト食道から筋線維芽細胞の単離

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

ヒトケロイド組織からの初代皮膚線維芽細胞の単離、培養、および特性評価

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...