この作品は（HPL）は、NIH助成金R01 GM087609、（MTL）は（HPL）にアンソニー・シュウ、NIHのグラントU01のHL099776に敬意を表しイングリッド・ライ、ビルシュウからの贈り物、Hagey研究所のための助成金によって部分的にサポートされていました小児再生医療とオーク財団（MTLに、GCGおよびHPL）。 GGWは医学のスタンフォードスクール、スタンフォード医学者研修プログラム、およびNIGMS訓練助成金GM07365によってサポートされていました。 ZNMは整形外科財団研究員助成とHageyファミリー基金によってサポートされていました。 MSHは再生医療のためのカリフォルニア工科大学（CIRM）臨床フェロー研修助成金TG2-01159、顎顔面外科学会（ASMS）/顎顔面外科財団（MSF）研究助成賞、および移植とティッシュ・エンジニアリングフェローシップ賞によってサポートされていました。

このプロトコルは、実験動物管理のスタンフォード大学紛争処理パネルにより承認された方法に従います。 マウス真皮の1消化<ol><li>ケタミンを100mg / kgのキシラジン+ 20ミリグラム/ kgのアセプロマジン+ 3 10mg / kgの腹腔内注射による麻酔後に頸椎脱臼によりマウスを安楽死させます。 注意：様々な年齢や背景を使用することができます。 </li><li>背部皮膚を剃ると毛を抜きます。約100,000個の細胞は、背側の皮膚の一片×100mmの60ミリメートルから単離することができます。 </li><li> 70％エタノールでマウスを水没し、乾燥さ清潔、無菌面に置きます。 </li><li>すぐに無菌解剖ハサミを使用して、背側マウスの皮膚を収穫。雌マウスでは、乳房組織を含めないようにしてください。 </li><li>尾の付け根から開始して、10トンまでの皮膚のに鉗子を使用し、超筋膜面に沿って切開する前に、横方向のカットを行います。 </li><li>収穫しながら、任意の皮下脂肪を含め慎重に避けます肌。任意の皮下脂肪のために採取した皮膚を調べ、慎重にメスの鈍いエッジを使用してそれを削り取ります。 </li><li>氷上でPBS洗浄を5倍に続いてベタジンで収穫皮膚を洗い流します。 注：これは、汚染を回避することができる無菌の近くの皮膚を維持することが重要です。 </li><li>かみそりの刃を使用して、サンプル2-3ミリの作品との均一な一貫性になるまで滅菌皿にはさみを解剖皮膚をみじん切り。 </li><li> DMEM中1mg / mlの濃度で50ミリリットルを20ミリリットルのコラゲナーゼIVを含むコニカルチューブを準備します。チューブあたり5匹のマウスに基づいてチューブに真皮を分割します。 </li><li>水浴またはオーブンのいずれかで37℃で1時間インキュベートしながら、積極的にサンプルを攪拌。 </li><li>インキュベーターからサンプルを取り出して、無菌フード内で針3-5xずに10ミリリットルの注射器を通過します。 </li><li> 37℃でバックインキュベーターにサンプルを配置し、さらに30分間激しく振ります。 </li><li>無菌フードに10mlのピペットを用いてピペット上下3-5xサンプル。新しい50mlコニカルチューブに100μmのフィルターを通して試料をピペット。 </li><li>細胞収率を最大化し、40ミリリットルの全量を同じフィルターを通して、10％FBSを含むDMEMの20ミリリットルを渡します。 4℃で8分間300gで遠心分離します。 </li><li>最初前残りの上清に、上部の脂肪層を除去するために細心の注意を取って、滅菌ガラスピペットを用いて上清を除去します。 注：このステップは、脂肪細胞の汚染を低減することが重要です。 </li><li> 20ミリリットルの10％FBS DMEM中でペレットを再懸濁します。 </li><li> 70μmのフィルターを通して細胞/ DMEM懸濁液を渡します。 </li><li> 10ミリリットル、10％FBS DMEMでフィルターを洗浄し、4℃で8分間300グラムで濾過サスペンションを遠心します。 </li><li>再び第1残りの脂肪層を除去するために世話をして、滅菌ガラスピペットを用いて上清を除去します。 </li><li>重要なRBC汚染がある場合（ペレットが目に見えて赤である）、ペレットを再懸濁20 mlのACK溶解緩衝液中で室温で5分間インキュベートします。それ以外の場合は24に進みます。 </li><li> FACS緩衝液（PBS、10％FBS、0.1％アジ化ナトリウム）の等量（20 ml）を追加し、その後混合し、非染色コントロールとして確保5mLのアリコートを維持します。 4℃で8分間300gで、残りのサンプルを遠心します。 </li><li>上清を除去し、氷の上にペレットを入れました。 FACS時間が利用できない場合、細胞は、この時点で凍結してもよいです。 </li></ol> FACSによる線維芽細胞の単離2。 <ol><li>各ペレットの系統抗体インキュベーションミックスの500μLを加えます。第1のチューブにDNアーゼ（10μg/ ml）を含むFACS緩衝液475μLを加え、その後、（1：100）、フルオロフォアコンジュゲートCD31を追加することでこれを行い、CD45（1：200）、Tie2の（1：50）、テル-119（1：200）、およびEpCAMの（1：100）抗体は、各抗体に対するそれぞれの希釈を達成します。 </li><li>系統抗体インキュベーションミックス500μlの各ペレットを再懸濁し、20分間氷上でこの懸濁液をインキュベートします。 </li><li> 5を追加サンプルにDNアーゼ（10μg/ ml）を含む、穏やかに混合mlのFACSバッファー。 4℃で8分間300gで遠心分離します。 </li><li>上清を除去し、ステップ26と同様の条件を用いて、DNアーゼ（10μg/ ml）および遠心分離を含む5ミリリットルFACS緩衝液で細胞ペレットを洗浄します。 </li><li> DNアーゼ（10μg/ ml）を含む500μlのFACS緩衝液中にペレットを再懸濁し、生存率染料コントロールとして確保50μlのアリコートを入れました。 </li><li>選択された染料のために示されている濃度の残りのサンプルに選択の生存率染料を追加します。 </li><li> FACS分析31を行い、生存率染料/ CD31- / CD45 - / Tie2- /テル-119- / EpCAM-細胞（ 図2A参照 ）のソート。ソート直接FACS緩衝液に。 </li></ol>

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</ol>

The authors have nothing to disclose.

この原稿に記載されているプロトコルは、亜集団のために選択するか、その後の分析の前に、細胞培養時間を必要とするいずれかの既存の方法に比較して、FACSベースの選別により、線維芽細胞を単離するための手段を提供しています。線維芽細胞のソートに皮膚の収穫からの所要時間は約6時間です。しかし、収穫に使用したマウスの数は、この推定値に影響を与えます。 <p class="jove_conten...

線維芽細胞は、しばしば、プラスチック基板に接着する紡錘状細胞と形態学的に定義されています。線維芽細胞は、胚および成体の臓器1における細胞外マトリックスの合成と改造を担当する原則細胞型です。線維芽細胞は、哺乳類の発生することが重要であり、各組織や臓器に存在する隣接セルの種類の動作に影響を与える細胞外環境に大きく貢献しています。 線維芽細胞はまた、膨大な臨床負担を引き起こす医学的条件の多様なセットの背後の主要な細胞型です。病理学的線維芽細胞の活性は、正常組織の機能を損ない、皮膚の創傷治癒、アテローム性動脈硬化症、全身性硬化症、および血管損傷2-5後のアテローム斑の形成後の組織及び（例えば肺や肝臓など）臓器線維症、瘢痕化が含まれます。特に、創傷治癒は、両方のは、急性および慢性的に、Dを含み、そのことを周囲の正常組織のようなどちらも似ているにも機能し、多様な病理学的状態全体でかなりの罹患率につながる瘢痕組織のeposition。損傷後、次に、構造ECM成分を分泌する細胞型に隣接パラクリン効果を発揮し、瘢痕組織6を堆積させることによって機械的安定性を復元する筋線維芽細胞への線維芽細胞の移行があります。 皮膚組織の発達時間全体の傷の修復の質と解剖学的部位との間に有意な変動が存在します。人生の最初の2つの学期では胎児が瘢痕化せずに治癒します。しかし、上と成人期を通じて第三期から、傷を癒す人間。部位特異的な、年齢別に加えて、創傷治癒の違いが存在します。皮膚創傷内の瘢痕組織沈着が9重要である一方で、口腔内の傷は、最小限の瘢痕形成7,8を改造します。論争は、cを持続します年齢や場所10,11の両方に関しては、創傷治癒の結果に地元の線維芽細胞の固有の特性に対する環境の相対的な影響をoncerning。皮膚の真皮およびそれ以前の胚（E15）対以降の胚（E18）真皮対経口マウスの治癒に大きな違いを考えると、それは特定の発達年齢で、様々な解剖学的部位の中の線維芽細胞の集団における本質的な違いが存在している可能性があります。 1986年、ハロルドF.ドヴォルザークは、腫瘍が12を治癒しない傷ある仮定しました。ドヴォルザークは、腫瘍が体に傷のように動作し、ホストの創傷治癒応答を活性化することにより、それらの間質を誘導することを結論付けました。多くの研究があるため、癌腫13-15の進行への線維芽細胞の寄与を調べたが、創傷治癒、アイデンティティと皮膚のCAの間質コンパートメントに貢献する線維芽細胞の胚起源の場合のようにしていますrcinomasは十分に定義されていません。この質問への答えは、抗癌治療のための潜在的に16有効な標的として腫瘍関連線維芽細胞を曝露する最近の研究で与えられた医学的関連を負いません。 特定と将来を見越して、生体内で線維形成の可能性に恵まれ、線維芽細胞系統の単離を効果的に急性および慢性疾患状態の広い範囲にわたって損傷に対するそれらの応答を操作に向けて不可欠なステップです。 1987年には、コーマックは乳頭と真皮網状層17,18内の1内に存在する線維芽細胞の2亜集団、1つを示しました。三亜集団は、卵胞19,20の乳頭地域の毛包に関連する発見されました。培養した場合、これらの線維芽細胞サブタイプの展示成長性の違い、形態、および成長因子/サイトカインは21-24プロファイル 。 現在までに、研究では、線維芽細胞、彼を調べますterogeneityは、主に適切に生体内での線維芽細胞間の発達と機能的多様性を特徴づけるために失敗しています。これは、部分的には、培養線維芽細胞集団への依存ではなく、すべての線維芽細胞25で表される自己表面レセプターに基づいて、細胞培養または正の選択の均一化効果の結果です。私たちの研究室からの最近の研究では、深遠な表面マーカーと、この原稿26で提示FACSに基づく分離方法によって単離された未培養線維芽細胞対培養において転写シフトを示しました。 その後、我々は、マウスの背側真皮内の特定の線維芽細胞系統を同定し、 エングレイルド-1の胚式で定義されたこの系統は、背部皮膚における結合組織沈着の主な原因であることを決定しました。創傷治癒、癌間質形成を含む線維症の急性および慢性型の両方の間の系統機能、放射線は、線維症27を誘導しました。明確な線維芽細胞系統の特徴付けは、線維形成の動作を調節することを目的と治療のための重要な意味を持っています。 むしろ細胞単離28,29を達成するために、in vitroでの操作に依存している既存のプロトコルを使用するよりも、ここでは詳述収穫プロトコル（ 図1）は、より正確に生体内での表現型と行動を捕獲線維芽細胞の収量有益な分析をするのに役立ちます。

<table><tbody><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>Elastase</td><td>Abcam</td><td>ab95133</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>A14430-01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) lysing buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>100 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>CD31</td><td>BioLegend</td><td>102421</td><td></td></tr><tr><td>CD45</td><td>BioLegend</td><td>103125</td><td></td></tr><tr><td>Tie2</td><td>BioLegend</td><td>124005</td><td></td></tr><tr><td>Ter-119</td><td>BioLegend</td><td>116233</td><td></td></tr><tr><td>EpCAM (CD326)</td><td>eBioscience</td><td>48-5791</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>propidium iodide (PI viability stain)</td><td>BioLegend</td><td>421301</td><td></td></tr></tbody></table>

murine dermal fibroblast isolation by facs

線維芽細胞は、細胞外マトリックスを分泌する責任原則細胞型であり、多くの臓器や組織の重要なコンポーネントです。線維芽細胞の生理学および病理学は、複数の器官における線維症、肥厚性瘢痕以下の火傷、虚血後の心機能の喪失、および癌間質の形成を含む臨床実体、のスペクトルを基礎となっています。しかし、線維芽細胞は、主として、その固有の不均一性、細胞の特徴付けが乏しいタイプ残ります。線維芽細胞の単離のための既存の方法は、深く細胞表現型と動作に影響を与える細胞培養の時間を要します。その結果、線維芽細胞の生物学を研究する多くの研究は 、in vitroでの操作に依存していると正確に生体内で線維芽細胞の挙動を捕捉しません。この問題を克服するために、我々はpreserviそれによって、細胞培養を必要とせず、成体マウスの背部皮膚からの線維芽細胞を単離するためのFACSベースのプロトコルを開発しました生理的な転写および各セルのプロテオミクスプロファイルをngの。特定の表面マーカーを発現する線維芽細胞の亜集団の事前選択または濃縮を回避し、この異機種全体で可能な限り包括的であることをむしろポジティブ選択戦略よりも-私たちの戦略は、系統陰性ゲート（林）を介して非間葉系統の排除を可能にしますセルタイプ。

このアプローチ（ 図1）の有効性は、我々の最近の刊行物27に詳細に検討することができる多くの方法で確認されました。これらは、ソートされた細胞と細胞塊の免疫細胞化学と新鮮にソートされた細胞の単一細胞の転写の分析が含まれます。線維芽細胞のソートではなく、直接、より正確文化に頼ることは、それらのインビボ表現型をキャプチャします。系統陰性枯渇のアプロ?...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

FACSによるマウス皮膚線維芽細胞の分離

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.

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

murine-dermal-fibroblast-isolation-by-facs

Research

JoVE Journal

Developmental Biology

21.0K ビュー.  Stanford University School of Medicine. 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.

ビデオ: Murine Dermal Fibroblast Isolation by FACS

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

発生生物学

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

原発性マウスメラノサイトと線維芽細胞培養の急速な生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

がん研究

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

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACSによる無動および損傷マウス骨格筋からの線維脂肪原性前駆細胞および筋幹細胞の単離および培養

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

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 Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

蛍光活性化細胞選別（FACS）によって新鮮な組織からの正常および癌関連線維芽細胞の分離

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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 ...

医学

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 ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.