著者らは、広範な議論のために、すべてのラボのメンバーに感謝の意を表したいとテクニックは、このプロトコルに対応しています。実験室で行われた研究は、科学技術省からの助成金によってサポートされていました（MOST 103から2314-B-002から126とMOST 102から2313-B-002から026-MY3）とトップ大学の計画のねらいからの助成金によって、国立大学、台湾の（104R350144）。

<ol>
	<li>Farese, R. V., Zechner, R., Newgard, C. B., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Problem+of+Establishing+Relationships+between+Hepatic+Steatosis+and+Hepatic+Insulin+Resistance.">The Problem of Establishing Relationships between Hepatic Steatosis and Hepatic Insulin Resistance.</a> Cell Metab. 15, 570-573 (2012).</li><li>Taubes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+research.+Unraveling+the+obesity-cancer+connection.">Cancer research. Unraveling the obesity-cancer connection.</a> Science. 335 (28), 30-32 (2012).</li><li>Apovian, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+adipocyte+formation.">Transcriptional control of adipocyte formation.</a> Cell Metabolism. 4, 263-273 (2006).</li><li>Aune, U. L., Ruiz, L., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Differentiation+of+Stromal+Vascular+Cells+to+Beige/Brite+Cells.">Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells.</a> J Vis Exp. , (2013).</li><li>Hsu, J. M., Ding, S. 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The authors have nothing to disclose.

ここでは、pADSCの初代細胞培養における脂肪組織の発達を研究するための信頼性の高いセルラーシステムを提案します。他の不死化細胞株と比較して、この方法は、脂肪細胞の分化プロセスまたはインビボで動物の開発に関連する他の間葉系統を研究するために適用することができ、高品質の成人間葉系幹細胞を大量に単離するための便利な方法を提供します。このプロトコルで重要な変更されたステップは、古い豚と19,20他の種に類似に比べて小さい子豚を処理するために容易であるため、我々は、7〜9日齢の子豚を使用してpADSCの収率および多分化をpADSCを導き出すことです豚の年齢21に減少します。 潜在的な幹細胞の供給源は、胚性幹細胞（ESC）、人工多能性幹細胞（IPSC）、および出生後の成体幹細胞を含みます。成体多能性幹細胞として分類ADSCの制約は、成体幹細胞の多分あります発散系統を分化にESCまたはIPSCと比較して相対的に限定されています。しかし、IPSCのESCと発癌特性の導出に関する倫理的な問題は、ESCとIPSC 22,23の適用を抑えます。したがって、多数の研究者らは、多能性を高めるために努力して成体幹細胞に焦点を当てています。長い間研究されてきた成体間葉系幹細胞（MSC）の最も一般的な供給源は、骨髄由来間葉系幹細胞24です。しかし、収穫骨髄は比較的痛みを伴う手順であると考えられます。別の問題は、骨髄からの幹細胞の収量が有限であることです。骨髄吸引物は、1mlあたり6×10 6有核細胞の平均を得て、MSCは、すべての有核細胞の0.001〜0.01％を表します。これらの欠点を検討した後、ADSCは、多能性幹細胞25,26を得るために、目立たない源として提案されています。 regeneraでADSCの使用に関する制限事項的な医学細胞収量と品質に大きく依存しています。したがって、このプロトコルでADSCを分離するために、ブタを用いる意義は、高品質の成体幹細胞を大量に生成するためです。豚が原因種27-30の間に匹敵する器官の大きさと多くの生理学的および生化学的類似性の人間を表す有用な動物モデルです。営利企業からhADSCを獲得することは高価であり、多くの場合、細胞は、操作継代または凍結保存されてきました。ヒト臨床サンプルを取得することがあるため倫理的な問題では比較的困難であるとADSCの生産が制限されています。私たちは、コラゲナーゼ消化後グラムの脂肪あたり約6×10 5 hADSCを導き出します。女性の乳房の脂肪組織（平均サンプリング）100gを、6×10 7個の細胞の合計を回収することができます。個々のマウスを使用して、収率はさらに制限されます。 1×10 6個の細胞の合計は、皮下マウスiの0.4グラムから収穫することができます大人のFVBマウスの両足からnguinal脂肪組織（6〜8週齢）。しかし、一人の個人豚（7〜9日齢）に、2×10 8個の細胞の合計を簡単に背脂肪デポから得られた皮下脂肪組織の60グラムから収穫することができます。このプロトコルで派生pADSCはフル間葉系多能し、適切な間葉系幹細胞マーカーを持っています。したがって、pADSCは、幹細胞の品質を損なうことなく、成体幹細胞を大量に得るための良好な供給源です。 pADSCの適用は、脂肪生成及び脂質生成などの脂肪細胞分化を解読に限定されるものではありません。最近では、ADSCは、再生医療22,31,32の分野では、幹細胞の人気の源となっています。他の幹細胞源に比べて、ADSCは、容易にアクセス可能と豊富であるというユニークな利点を保持し、その堅牢な多分化は、幹細胞治療および組織電子のための有望な源であることが実証されていますngineering 22,33,34。脂肪組織の容易なアクセス性がADSC多能性前駆細胞を得るために少なくとも侵入的な方法の一つになります。最近、我々はpADSCは間葉分化（未発表データ）に限定されないことを示す、グルコース応答性インスリン分泌クラスタにpADSCを分化しました。その他にも、ADSCは、このような（hADSC 37またはpADSC 38から）（hADSC 35またはpADSC 36）から内胚葉肝細胞または外胚葉の神経細胞などの他の胚葉由来の多くの細胞型に分化させることができることが実証されてきました。したがって、pADSCは、所望の系統を得るために発散分化過程に細胞を誘導することによって、ハイスループット薬物または生体物質のスクリーニングに使用することができます。したがって、このプロトコルで派生pADSCは、再生医療研究のための幹細胞治療および組織移植における潜在的なアプリケーションを持っています。

米国の人口の約30％に存在する肥満は、30以上のボディ・マス・インデックスで、世界的に流行している現象1として浮上しています。肥満は心血管疾患、2型糖尿病、および癌2-4を含む関連合併症を引き起こす傾向があります。そのため、肥満に対処することは重要な課題です。肥満は、脂肪組織の大規模な拡張によって明らかにされており、現代社会における過剰な食物消費と座りがちな生活習慣に起因します。したがって、脂肪生成及び脂質生成の転写調節を解読することは、肥満や糖尿病の5を治療するための約束を保持することができます。 3T3-L1、3T3-F442Aおよび他のマウス脂肪生成細胞株は、脂肪組織の開発中に脂肪生成または脂質生成を研究するために適用されています。しかし、 インビボ 6 インビトロおよび動物の細胞株との間の調節機構に何らかの不一致があります。プライマリ脂肪-DERI間質血管細胞画分におけるVED幹細胞（ADSC）は、白色脂肪組織から直接単離され、分化するように誘導することができます。脂肪細胞へのADSCの分化は、最も可能性の高い生体内 7 における脂肪組織開発における脂肪生成及び脂質生成の過程を再現します。 ブタは、脂肪組織の発達に脂肪生成及び脂質生成を研究するための適切な動物モデルです。我々の以前のブタ研究8-10ステロール調節エレメント結合転写因子1C（SREBP1c）、脂質合成脂肪酸シンターゼの転写を調節することが知られている重要な転写因子の発現は、ブタ肝臓における多価不飽和脂肪酸（PUFA）によって阻害されることを実証しますそして、脂肪組織。 インビボおよびインビトロでの PUFAの減少ブタSREBP1cの発現は、ヒトおよびマウス11〜13のような他の種と同様です。 in vitroでのこれらのブタの研究は、差分に主にブタADSC（pADSC）由来の脂肪細胞をerentiated。したがって、pADSCこの初代細胞培養物は、脂肪組織の発達または他の幹細胞の用途を研究するための信頼性のあるセルラーシステムとして機能するように使用することができます。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase, Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995-040</td><td></td></tr><tr><td>DMEM/F-12, HEPES</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Biological Industries</td><td>04-001-1&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-Streptomycin-Amphotericin B (P/S/A) solution</td><td>Biological Industries</td><td>03-033-1</td><td>For antibiotics and antimycotic usage</td></tr><tr><td>&amp;alpha;MEM, no nucleosides&amp;nbsp;</td><td>Life Technologies</td><td>12561-049</td><td></td></tr><tr><td>ACK lysis buffer&amp;nbsp;</td><td>Life Technologies</td><td>A10492-01</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Life Technologies</td><td>25200072</td><td></td></tr><tr><td>CD4a-PE</td><td>Sigma-Aldrich</td><td>SAB4700063</td><td></td></tr><tr><td>CD29-PE</td><td>Sigma-Aldrich</td><td>SAB4700398</td><td></td></tr><tr><td>CD31-PE</td><td>Sigma-Aldrich</td><td>SAB4700467</td><td></td></tr><tr><td>CD44-PE</td><td>Sigma-Aldrich</td><td>SAB4700183</td><td></td></tr><tr><td>CD45-PE</td><td>Sigma-Aldrich</td><td>SAB4700483</td><td></td></tr><tr><td>CD90-PE</td><td>Sigma-Aldrich</td><td>SAB4700686</td><td></td></tr><tr><td>HLA Class I-PE (MHC I)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SAB4700640</td><td></td></tr><tr><td>HLA-DR-PE (MHC II)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SAB4700662</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>3,3&#039;,5-Triiodo-L-thyronine (T3)</td><td>Sigma-Aldrich</td><td>T6397</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T2036</td><td></td></tr><tr><td>3-isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich</td><td>I7018</td><td></td></tr><tr><td>Dexamethasone&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Rosiglitazone</td><td>Cayman</td><td>71740</td><td></td></tr><tr><td>&amp;beta;-Glycerophosphate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G9422</td><td></td></tr><tr><td>2-Phospho-L-ascorbic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>49752</td><td></td></tr><tr><td>TGFB1 Recombinant Human Protein&amp;nbsp;</td><td>R&amp;amp;D Systems&amp;nbsp;</td><td>240-B-002</td><td></td></tr><tr><td>Oil Red O&amp;nbsp;</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich</td><td>A5533</td><td></td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>198161</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carbon Steel Blades</td><td>Thomas Scientific</td><td>6727C18</td><td></td></tr><tr><td>Falcon 100 &amp;micro;m cell strainer</td><td>Corning&amp;nbsp;</td><td>352360</td><td></td></tr><tr><td>Falcon 6-well plate</td><td>Corning&amp;nbsp;</td><td>353046</td><td></td></tr><tr><td>Falcon 100 mm&amp;nbsp; dish</td><td>Corning&amp;nbsp;</td><td>353003</td><td></td></tr></tbody></table>

isolation and differentiation of adipose-derived stem cells from porcine subcutaneous adipose tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

豚の背側皮下脂肪由来pADSCは、培養プレートまたはディッシュに播種し、 図2に示した。間質-血管画分由来pADSCの形態は、マウスまたはヒトADSCに似ています。播種後二十四時間、サブコンpADSCが付着し、拡張線維芽細胞様の形態（ 図2A）を有しています。 pADSCは、72時間以内にコンフルエントになると脂肪細胞または他の間葉型分化（ 図2B）の準備ができているでしょう。化学誘導および成熟脂肪細胞が脂肪細胞への分化（ 図2C）を示すpADSCsの90％以上と分化の9日後に観察することができた後に強力な脂肪生成潜在能力を発揮pADSC。 このプロトコルで導出pADSCの特性に対処するために、pADSCの表面マーカーは、肛門、フローサイトメトリーによって評価しました。ysis。 図3に示すように、CD29、CD44、CD90、およびMHC I（またはHLA I）を含む間葉系幹細胞のための表面マーカーは、高度に発現されました。このようなCD4a、CD31、CD45およびMHC II（またはHLA II）のような負の表面マーカーは、プロトコル（ 図3）で求めたpADSCでかろうじて検出可能でした。これらの結果は、骨髄やリンパ系前駆細胞を含む、重要な内皮細胞または造血幹細胞の混入のないこれらのpADSC展示間葉系幹細胞の特性ことを示しています。 さらにそのpADSCを確認するために、間葉系幹細胞を表し、pADSCの多能は、脂肪細胞、骨細胞および軟骨細胞への分化によって調べました。これらの脂肪細胞、骨細胞および軟骨細胞は、特定の染料、オイルレッドO、アリザリンレッドS、およびトルイジンブルーO、それぞれ（ 図4）によって染色しました。これらのデータは、このプロトコルがムーを保持pADSCを生成することを示します間葉系前駆細胞に似た完全な特性を持つltipotency。 <img alt="図2" src="/files/ftp_upload/53886/53886fig2.jpg" /> ブタの背脂肪地域からpADSCの図2形態。（A）サブコンフルエントpADSCが付着し、6ウェル培養プレートに24時間播種した後に拡大しました。 （B）pADSCは6ウェル培養プレートに72時間播種後コンフルエントになりました。 （C）成熟脂肪細胞をpADSCから脂肪細胞への分化の9日後に観察しました。画像は位相差顕微鏡を用いて100倍の倍率で撮影した。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53886/53886fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53886/53886fig3.jpg" /> 幹細胞の図3.同定pADSCための表面マーカー1×10 5 pADSCの特異的抗体と反応させ、フローサイトメトリー分析により、幹細胞マーカーについて分析しました。数値は、染色していない対照と比較して人口（赤）で染色された細胞の割合を示しています。 x軸は相対蛍光強度を表します。 y軸は細胞の集団を表す。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53886/53886fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53886/53886fig4.jpg" /> pADSCのpADSC。多能の図4.多能分化は、（A）脂肪細胞、（B）骨（C）軟骨細胞にpADSCを微分することによって決定され、それぞれの代表的な染料、オイルレッドO、アリザリンレッドS、およびトルイジンブルーOで染色しました。いまGESはそれぞれ、位相差顕微鏡を用いて、（A）100×（B）200×、および（C）100倍の倍率で撮影した<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53886/53886fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues at JoVE.com

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

isolation-differentiation-adipose-derived-stem-cells-from-porcine

Research

JoVE Journal

Developmental Biology

29.1K ビュー.  National Taiwan University. This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies. 

ビデオ: Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

インスリン産生細胞への分化のためのラット脂肪組織間葉系幹細胞の単離

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

発生生物学

Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine branches, particularly cell therapy, are novel approaches to improve and restore the urethral sphincter function. Even though injection of active functional cells is routinely performed in clinical settings by needle and syringe, these approaches have significant disadvantages and limitations. In this context, needle-free waterjet (WJ) technology is a feasible and innovative method that can inject viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we used WJ to deliver porcine adipose tissue-derived stromal cells (pADSCs) into cadaveric urethral tissue and subsequently investigated the effect of WJ delivery on cell yield and viability. We also assessed the biomechanical features (i.e., elasticity) by atomic force microscopy (AFM) measurements. We showed that WJ delivered pADSCs were significantly reduced in their cellular elasticity. The viability was significantly lower compared to controls but is still above 80%.

We present a method of cell injection via needle free waterjet technology coupled with a sequela of post-delivery investigations in terms of cellular viability, proliferation, and elasticity measurements.

ウォータージェット技術によるブタ脂肪組織由来間質細胞の注入

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine ...

生物工学

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

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 ...

医学

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 ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

アカゲザル脂肪由来幹細胞の単離・増殖・分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

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 ...

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

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インスリン産生細胞への分化のためのラット脂肪組織間葉系幹細胞の単離

ウォータージェット技術によるブタ脂肪組織由来間質細胞の注入

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

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

アカゲザル脂肪由来幹細胞の単離・増殖・分化

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