Name: immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)
Uploaded: 2016-08-11T14:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

この作品は、カンナビノール（THC）はNIH DK095137によってサポートされています。私たちは、記載された方法の開発と高度化に貢献して現在および過去の研究室のメンバーに感謝します。

倫理声明：動物の使用およびケアに関するミシガン大学委員会（UCUCA）は、実験動物の管理と使用に関する指針（実験動物研究所、国家研究評議会）に従い、すべての方法およびプロトコルを承認しました。マウスは、ミシガン大学の飼育に維持され、食料と水を自由に与え、12時間の明/暗サイクルで維持されています。 1.準備<ol><li> DMEM、10％FBS、1×P / S / G、および1×抗生物質/抗真菌剤で初代培養培地を準備します。これは、消化の前に組織を維持するために使用されます。 37ºCの水浴中にストックし、アリコートを置きます。 </li><li> 5匹までの、単離される脂肪パッドの種類ごとにHBSS（+カルシウム、+ Mg）を中に溶解させミリリットルの5mg /でタイプIIIコラゲナーゼ溶液の5ミリリットルを準備します。 10ミリリットルを作る、単一の遺伝子型からSQとVISを分離する際に、例えば、野生型および変異体SQとVIS場合、20ミリリットルを作ります。 7.4およびSTEにpHを調整しますフィルタを波立たせます。 50ミリリットルチューブに小分けし。光を避けて脇に置きます。 </li><li>手術野を消毒するために70％エタノールを使用してください。拭うと青のパッドでカバーしています。青いパッドの上にいくつかのエタノールをスプレーしてください。 </li><li>発泡スチロールの板に吸収パッドを突き止めるし、はさみでトリミングするために22 G針を使用してください。青いパッドの上にボードを設定し、解剖を開始するまで、別の青色のパッドでカバーした後、エタノールでパッドをスプレーします。 </li><li>手術野に隣接するスタンドにエタノールと場所を持つ2つの50mlチューブを埋めます。エタノールにハサミやピンセットを置きます。 </li><li>手術器具をエタノールを洗い流すためにPBSプラス1×抗抗持つ別の50ミリリットルチューブを埋めます。 </li><li>フードでは、各組織型のラベル60ミリメートルの料理とは、37ºCに温めた培養培地で満たします。手術領域に隣接するプレートを置きます。 </li></ol>皮下（SQ）脂肪パッドの2の単離<ol><li>イソフルランおよび気胸の過剰投与によりマウスを安楽死させます。 </li><li>優しく70％エタノールでマウスをスプレーし、仰臥位横たわっていました。 22 G針で発泡ボードに足をピン。 </li><li>下腹部の皮膚の小さなカットを行います。鉗子でカットの上部を持ち、はさみを取り、腹膜から皮膚を分離します。 </li><li>皮膚が胸部に腹部から腹膜から分離された後、本体の側面に沿って皮膚の横カットを行うことで、離れて頭に向かって腹膜から肌を反映しています。 </li><li>体から離れ鼠径部脂肪を保持し、残りの皮膚を反映し、22 G針でボードに突き止めます。 </li><li>細かいハサミやピンセットに切り替えます。腹膜の近くに原点にピンセットで鼠径部脂肪パッドをつかみ、皮膚とのSQを汚染回避鼠径部に向かって進行し、皮膚や鼠径部脂肪の間離れてスニップ。 </li><li>ラベルされた60ミリメートル皿にばらばら鼠径部脂肪パッドを配置します。 </li></ol>内臓（VIS）脂肪パッドの3分離<ol><li> 2.5ステップと同様に、内臓脂肪パッドおよび腸を露出させる開放腹膜を切りました。 </li><li>胸部に向かって腸を離れて移動します。 </li><li>遠位端にVIS脂肪パッドをつかみ、ゆっくりと引き上げます。組織（雌マウスを使用している場合、または子宮）精巣上体を除外するように注意しながらVIS脂肪パッドを解剖。 </li><li>ラベルされた皿、残りVISパッドの繰り返しステップ3.3で配置します。 </li></ol>脂肪パッドの4コラゲナーゼ消化<ol><li>組織培養フードに60ミリメートルの皿を移動し、メディアを吸引します。 </li><li>湾曲したハサミで脂肪パッドをミンチ後、コラゲナーゼ溶液に追加します。エタノールおよびPBSで組織タイプの間にはさみとピンセットを洗浄してください。 </li><li>組織が消化されるまで、300 rpmで10〜20分間、37ºCで振ります。 SQのいくつかの作品がまだ表示されている場合は、許容可能です。これは、後の手順で対処します。 </li><li> collagを停止するためにコラゲナーゼ溶液を培地25 mlを加え消化された組織を分散させるために10ミリリットルの血清学的ピペットを用いenase処理、ピペットを上下に10回。 </li><li> 100μmの細胞ストレーナーを用いて株細胞。 300×gで10分間遠心分離します。 </li><li>慎重にペレットを乱さし、細胞を懸濁するペレットと静かにピペットに滅菌水5ミリリットルを追加していないメディアをオフにデカント。赤血球を溶解するために2分を待ちます。 </li><li>新しい100μmのセルストレーナーを介して、10％FBSおよび株細胞と培地25ミリリットルを追加します。 </li><li> 300×gで10分間遠心分離します。デカントメディアと培養培地の1ミリリットルでペレットを再懸濁し。 </li><li> 1トリパンブルー：1を用いて、血球計数器を用いて細胞を数えます。 </li><li> 6ウェルプレートに1ウェル内のプレート1×10 6個の細胞。 </li></ol> 5.磁気細胞分離<ol><li>細胞接着の約4~6時間後、HBSS（-Ca、-Mg）で細胞を2回洗浄します。 0.05％トリプシンを用いて接着細胞を解離します。 1mLの分離緩衝液中でトリプシン細胞懸濁液を希釈し、は回転300×gで5分間。 </li><li>上清を吸引。 500μlの緩衝液中で細胞を再懸濁し。 10μlのアリコートを削除し、血球計数器を用いて細胞数を計測します。 </li><li> 300×gで5分間スピン細胞。 </li><li>完全に上清を吸引。 90μlの緩衝液中で細胞を再懸濁し。 10μlの抗のSca1-FITC一次抗体を追加します。よく混ぜ、暗所で4ºCで10分間インキュベートします。 </li><li> mlのバッファーと遠心セル300×gで5分間1で細胞を洗浄。完全に上清を取り除きます。 </li><li> 80μlの緩衝液中で細胞を再懸濁し。 20μlの抗FITCマイクロビーズを追加します。よく混ぜ、暗所で4ºCで15分間インキュベートします。 </li><li> mlのバッファーと遠心セル300×gで5分間1で細胞を洗浄。 </li><li>上清を除去し、500μlの緩衝液中にペレットを再懸濁。 </li><li>磁気ホルダーに列を配置し、500μlの緩衝液でカラムをすすぎます。 </li><li>カラムに細胞懸濁液を追加します。ピペッティングしながら気泡を避けてください。 </li><li>非標識のSca1を収集- &lt;/ SUP&gt;細胞および500μlの緩衝液でカラム3回洗浄します。リザーバが空である場合にのみ、新しいバッファを追加します。ピペッティングしながら気泡を避けてください。 </li><li>磁気ホルダーから列を削除し、15ミリリットルコニカルチューブに配置します。 </li><li>列にバッファリングし、カラムリザーバーを介して供給されるプランジャーを押してのSca1の+を溶出1ミリリットルを追加します。 </li><li> 300×gで5分間、細胞画分をスピンダウン。 1mLの培地中に再懸濁した細胞。 </li><li>標識し、非標識画分から10μlのアリコートを削除し、血球計数器を用いて細胞数を計測します。 </li><li>プレート6ウェルプレートの1ウェル中の各細胞画分。 </li></ol>フローサイトメトリーでのSca1 高のACSの免疫磁気分離の6.検証<ol><li> HBSS（-Ca、-Mg）で2回細胞を洗浄し、0.05％トリプシンを用いて細胞を解離します。 </li><li> 5分間、300×gで遠心分離した細胞。 1ミリリットル培養培地中で細胞を再懸濁し、血球計数器を用いてカウントします。 </li><li>入手&gt; 10 6 </SUP&gt; 1ミリリットル培養培地中の細胞と5分間、300×gで遠心。 </li><li>さらに2回上清を除去し、ステップ6.3を繰り返します。 </li><li> 2％ヤギ血清+ RTで30分間、2％BSA及びブロック1mlで再懸濁細胞。 </li><li> 5分間、300×gで遠心分離した細胞。 </li><li>ブロッキング溶液を除去します。 </li><li> 4℃で2％ヤギ血清および2％BSA + PBSで100μlのPBS中で、：または抗のSca1アレクサフルオロ647（4000.25μgの、1）：ラットIgG2aのアレクサフルオロ647（4000.25μgの、1）を追加します。暗所で30分間。 </li><li>細胞に冷PBSの1ミリリットルを追加します。 4°Cで5分間遠心分離300×gで。 </li><li>さらに2回上清を除去し、ステップ6.9を繰り返します。 </li><li> 1mlのPBSで細胞を再懸濁し。フローサイトメトリー分析の準備のために100μmのセルストレーナーを介して細胞懸濁液を渡します。 </li></ol>

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ここで我々は分離と異なる脂肪パッドからマウスのASCの免疫細胞分離およびin vitro実験のためのそれらの使用を実証します。提示された方法は、ASCを9,14の技術的に複雑で高価なFACS媒介分離よりも有利で ​​あるのSca1陽性のASCの多数の迅速な隔離のために有効です。 FACSは異なり、免疫細胞の分離は、標的細胞集団を同定するための多数の抗原の使用を許可しません。表面抗原...

細胞抗原1（のSca1、またはLy6A / E）の幹第造血および間葉系幹細胞5,6によって発現される細胞表面マーカーとして同定されました。マウス脂肪デポから得られた脂肪組織の間質の血管画分（SVF）は、線維芽細胞、マクロファージ、血管内皮細胞、神経細胞、および脂肪細胞前駆細胞の7を含む細胞の異種集団です。脂肪細胞の前駆細胞、または脂肪由来幹細胞（ASCには）、コラーゲンが豊富な血管周囲の細胞外マトリックス（ECM）8内に存在する非脂質を含んだ細胞です。 CD34 +：のSca1 + 9とCD29 + -約SVFの50％は、系統陰性（林）として特徴付けられるのASC、から構成されています。 ; in vitroでの脂肪細胞分化が可能である脂肪細胞前駆細胞、 - CD24：これらの細胞のほとんどはのSca1 +ですしかし、細胞の一部のみ（SVFの0.08％）がのSca1を構成します<sアップ&gt; +：増殖性およびin vivoの条件9で脂肪細胞に分化を完全に行うことができますCD24 +細胞。 CD24からCD24 +細胞を区別することなくのSca1 + SVFを使用しての潜在的な警告にもかかわらず-免疫細胞分離を使用して、貯蔵脂肪からのSca1 +のASCを、細胞を単離一次脂肪細胞前駆細胞の細胞自律的な表現型を決定するための効率的かつ実用的なアプローチです。 肥満および糖尿病の分野では、組織線維症及び炎症が2型糖尿病3の発達および維持において重要な役割を果たしています。最近、徳永ら 。 （VIS、または内臓）鼠径部（または皮下、SQ）およびperigonadal C57BL6 / J脂肪デポから分離されたのSca1 high細胞は、in vitro 10 で異なる遺伝子署名およびECMリモデリングを示すことが示されました。 MMP14（MT1-MMP）、膜 - トンの原型メンバーYPEマトリックスメタロプロテアーゼ（MMP）ファミリーは、コラーゲン分解活性の1を介して、白色脂肪組織（WAT）の開発を仲介します。 以下のプロトコルを使用して単離され、濃縮された細胞を用いて行ってもよいの実験例は、三次元培養、分化研究、コラーゲン分解アッセイ、およびRNA配列10,11を含みます。分解アッセイは、テロペプチド11,12の保全を確実にするために、酸抽出コラーゲンで行われるべきです。次のプロトコルが異なる脂肪デポからの一次血管間質細胞を分離し、免疫磁気細胞分離を用いて、脂肪細胞の前駆細胞を豊かにする方法を紹介します。細胞選別の有効性は、フローサイトメトリーを用いてとのSca1プロモーター 13によって駆動されるのSca1 +細胞においてGFPを発現するのSca1-GFPマウスを用いによって評価されます。

<table><tbody><tr><td>Type 3 Collagenase</td><td>Worthington Biochemical</td><td>LS004182</td><td>Tissue digestion</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>High-glucose culture medium</td></tr><tr><td>Pen/Strep/Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td>Media antibiotic</td></tr><tr><td>Anti-anti (100x)</td><td>Gibco</td><td>15240-062</td><td>Media antifungal</td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>PBS (1x, pH 7.4)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Trypsin (0.05%)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell strainer</td><td>BD Bioscience</td><td>352360</td><td>100-&amp;mu;m cell strainer</td></tr><tr><td>60 mm plates</td><td>BD Falcon</td><td>353004</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>14001-12</td><td>Large</td></tr><tr><td>Scissors</td><td>FST</td><td>14091-11</td><td>Fine, curved tip</td></tr><tr><td>Large Forceps</td><td>FST</td><td>11000-12</td><td></td></tr><tr><td>Fine Forceps</td><td>Any vendor</td><td></td><td></td></tr><tr><td>25G 5/8&amp;rdquo; needles</td><td>BD</td><td>305122</td><td></td></tr><tr><td>22G 1.5&amp;rdquo; needles</td><td>BD</td><td>305159</td><td></td></tr><tr><td>15 ml conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>50 ml conical tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>MACS separation columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Anti-Sca1 microbead kit (FITC)</td><td>Miltenyi Biotec</td><td>130-092-529</td><td>FITC-anti-Sca1 1&amp;ordm;Ab and anti-FITC microbeads 2&amp;ordm;Ab</td></tr><tr><td>AutoMACS running buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>MiniMACS separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Blue chux pads</td><td>Fisher</td><td>276-12424</td><td></td></tr><tr><td>Absorbent pads</td><td>Fisher</td><td>19-165-621</td><td></td></tr><tr><td>Styrofoam board</td><td></td><td></td><td>Use from 50 ml tubes</td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Rat IgG2a Alexa Fluor 647</td><td>Invitrogen</td><td>R2a21</td><td></td></tr><tr><td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td><td>Invitrogen</td><td>MSCA21</td><td></td></tr><tr><td>Rat IgG2a R-PE</td><td>Invitrogen</td><td>R2a04</td><td></td></tr><tr><td>Rat IgG2a anti-mouse F4/80 R-PE</td><td>Invitrogen</td><td>MF48004</td><td></td></tr><tr><td>Round-bottom tube</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>HBSS (&amp;ndash;Ca, &amp;ndash;Mg)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>HBSS (+Ca, +Mg)</td><td>Gibco</td><td>14025-092</td><td>For collagenase solution</td></tr><tr><td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td><td>Prepare in house&lt;sup&gt;12&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>10x MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>0.34 N NaOH</td><td>Prepare in house</td><td></td><td></td></tr><tr><td>Cover slips</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td><td>Invitrogen</td><td>A-20004</td><td></td></tr><tr><td>0.89 M NaHCO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>Gibco</td><td>25080-094</td><td></td></tr></tbody></table>

immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

別の脂肪パッドからのSca1 高のASCの濃縮。 SQ脂肪表示線維芽細胞などから単離された血管間質細胞は、関係なくのSca1の発現レベル（ 図1A）のセル形状を伸ばしました。一方、VIS（eWAT由来）のSca1 高いとのSca1 低細胞は、その細胞形状の明確な違いを示しています。 SQのように（IWAT由来...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪デポ固有のSca1の免疫磁気分離<sup&gt;高</sup&gt;脂肪由来幹細胞（ASCに）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

6.8K ビュー.  Biointerfaces Institute, University of Michigan. We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

ビデオ: Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

発生生物学

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神経紋の分離、培養、脂肪形成誘導、周食脂肪組織由来の脂肪由来幹細胞

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

マウス腹腔内脂肪貯蔵所からの脂肪形成性および線維炎症性間質細胞亜集団の単離

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

免疫・感染学

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

生物学

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

ヒト脂肪由来間葉系幹細胞の単離・増殖のための酵素フリー法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

医学

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

脂肪組織から間葉系幹細胞を得る技術および長期凍結保存における間質血管画分のキャラクタリゼーション

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

マウス上端体脂肪組織からの脂肪前駆細胞の調製

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

脂肪組織由来幹細胞の単離と特性評価

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

著者スポットライト:Sprague Dawleyラットの脂肪組織に由来する間葉系幹細胞の単離と同定  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

脂肪デポ固有のSca1の免疫磁気分離

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強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

神経紋の分離、培養、脂肪形成誘導、周食脂肪組織由来の脂肪由来幹細胞

マウス腹腔内脂肪貯蔵所からの脂肪形成性および線維炎症性間質細胞亜集団の単離

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

ヒト脂肪由来間葉系幹細胞の単離・増殖のための酵素フリー法

脂肪組織から間葉系幹細胞を得る技術および長期凍結保存における間質血管画分のキャラクタリゼーション

マウス上端体脂肪組織からの脂肪前駆細胞の調製

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定