私たちは、ジャニーン・ベッカー、キャロライン・ビッケルマンとルース・ニッケルズの優れた技術支援のために感謝しています。 （ - ドイツ研究協会DFG） - LA 2682 / 7-1この研究は、ドイツForschungsgemeinschaftの助成金によって賄われていました。

すべての手順は、実験動物の使用のための健康ガイドラインの国立研究所に従って実行し、施設のガイドライン（LandesamtエリーゼSoziales、GesundheitウントVerbraucherschutz、アプト。Lebensmittel-ウントVeterinärwesen、Zentralstelle、ザールブリュッケン、ドイツ）を追跡しました。 手術器具の調製<ol><li> 10％ウシ胎児血清（FCS）、100U / mLのペニシリン、0.1mg / mlのストレプトマイシン、準備解剖ハサミ、外科用鉗子、小さな調製はさみ、微細鉗子及び15mLのダルベッコ改変イーグル培地（DMEM滅菌ペトリ皿を保ちます）精巣上体脂肪パッドを収穫するため。 </li><li> 5分間消毒液に手術器具を公開します。あるいは、（; 121℃、20分間蒸気滅菌）それらを滅菌。 </li></ol> 2.動物および麻酔<ol><li>慎重に研究のために示されているように、マウスの系統を選択し、<html>調査中の対象。 注：本研究では、精巣上体脂肪の収穫のためのドナーとしてのオスの野生型C57BL / 6マウスを使用しました。興味深いのは、AD-MVFは、トランスジェニック緑色蛍光タンパク質（GFP）陽性ドナー動物（C57BL / 6-TG（CAG-EGFP）1Osb / J）22から単離することができます。これは、断片は、GFP陰性野生型マウス14への移植後のGFPの免疫組織化学染色によって容易に検出されている主な利点を負いません。 </li><li>キシラジン（15ミリグラム/ kg）およびケタミン（/ kgを75 mg）を腹腔内注射で動物を麻酔。動物が深く無応答とつま先のピンチを実行することにより、麻酔をかけていることを確認してください。ドナー動物の脂肪、収穫後に屠殺されているとして、目の潤滑剤が示されていません。 注：鎮痛および外科無菌性は、実験が計画されている国や機関のそれぞれのガイドラインと一致していることを確認してください。 </li></ol>精巣上体脂肪パッドのル &quot;&gt; 3。収穫<ol><li>操作テーブルに動物を転送します。手術用実体顕微鏡下仰臥位で動物を置き、つま先のピンチを使用して深い麻酔を確認。 </li><li>手術用ドレープにそれらをテーピングで足を固定化し、解決策を消毒し、腹部を消毒します。 </li><li>解剖ハサミで下の筋肉層のない腹部の皮膚を分離します。 </li><li>解剖ハサミで正中開腹術を実行し、横方向に腹壁のフラップを展開。 </li><li>両側精巣、精巣上体および微細鉗子（ 図1）を用いて、精巣上体脂肪パッドを識別する。 脂肪パッドの糞便汚染を防止するために、腸の構造に害を与えないでください。 </li><li>実体顕微鏡下の小さな準備ハサミと細かい鉗子で精巣上体脂肪パッドを収穫。 副睾丸脂肪の間に数mmの安全マージンを保ちます偶然の精巣上体収穫のリスクを減らします。 注：この手順は、実体顕微鏡なく行うことができます。しかし、これはさらに精巣上体および精索の不慮の損傷のリスクを高める可能性があります。 </li><li>細胞実験室への輸送のために、37℃で予備加熱DMEMの15 mLを含むペトリ皿に精巣上体脂肪パッドを転送します。 </li><li>腹部大動脈または頸椎脱臼を切開することによって動物を生け贄に捧げます。 </li></ol>広告-MVFの4分離<ol><li>リン酸緩衝生理食塩水（PBS）15mLの滅菌14-mLのポリプロピレン（PP）チューブ、滅菌50mLの三角フラスコ、滅菌した1.5 mLのコニカルマイクロ遠心チューブ、滅菌微細なハサミで三枚の滅菌ペトリ皿を準備します。 </li><li>層流フードの下でPBS 15mLでペトリ皿に三回脂肪パッドを洗浄します。 </li><li> 14-mLのポリプロピレンチューブに脂肪を移します。 Tによって（ML）に採取脂肪組織の量を決定します彼チューブスケール。均質な組織懸濁液が得られるまで微細はさみで機械的に脂肪組織をミンチ。 </li><li>ピペットを測定する10ミリリットルを用いて50 mLの三角フラスコにコラゲナーゼNB4G（0.5 U / mLのPBS）2つの容量と細分化した組織を移します。総量は、ステップ4.3で測定された脂肪組織の容積の3倍になります。 37℃、5％CO 2を含む加湿大気条件：自動攪拌機（25ミリメートル磁気攪拌棒のサイズ）によって激しく攪拌しながら10分間インキュベーター内組織消化を行います。 </li><li>消化を停止させることができるかどうかを判断するために顕微鏡下で消化された組織の小部分（10μL）を観察します。消化物は、主に酵素消化工程（ 図2）を停止するための適切なポイントを示し、次の単一細胞への「遊離」AD-MVFが含まれていることを確かめます。 注：この手順は、手順と経験を必要とし、品質のために重要です広告-MVFを単離しました。広告-MVFせずに、単一細胞懸濁液中の脂肪の消化結果を延長しました。 </li><li> PBS / 20％FCS 2つの容量を有する酵素を中和します。総体積は、ステップ4.4において、細胞容器懸濁液の体積の3倍となります。新しいPPチューブにバック細胞血管懸濁液を移します。 </li><li>重力によって脂肪残りからAD-MVFを分離するために37°Cで5分間懸濁液をインキュベートします。次いで、注意深く1-mLの精密ピペットで主脂肪上清を除去します。サスペンションは、無脂肪のように表示されるまで、100-μLの精密ピペットでこのサイクルを数回繰り返します。 </li><li> 50 mLのコニカル遠心管の上部に500μmのフィルターを置きます。残りの脂肪塊を除去するために濾過膜上に14-mLのPP管からの10-mLのメスピペットを用いて細胞血管懸濁液を移します。個々のAD-MVF計画実験のため分離の数に応じて新たな14-mLのポリプロピレンチューブに濾過した懸濁液を移します。 注：のためにvitrで微小血管ネットワークの形成に焦点を当てOアッセイは、さらに顕微鏡分析の間に結像品質を改善するために、細胞容器懸濁液を精製することが有益であり得ます。この目的のために、懸濁液をさらにフィルター上に集めAD-MVFから単一細胞を除去するために20μmのフィルターで一度ろ過してもよいです。 </li><li>遠心分離機AD-MVFを含むペレットを得るために、細胞容器懸濁液（600×gで、5分間、室温）。 </li><li> 1mLのが残されるまで遠心分離後、上清を除去します。この1mLのAD-MVFでペレットを再懸濁し、1.5 mLのコニカル遠心チューブに懸濁液を移します。 </li><li>遠心分離ペレットを得るためにマイクロチューブのAD-MVF懸濁液（600×gで、5分間）。 </li><li> PBS / 20％FCSの必要な最終体積でペレットを再懸濁し、上清を取り除きます。 注：分離株あたりの広告-MVFの数は、顕微鏡の計数によって評価することができます。この目的のために、最終細胞-vesseの1/10L懸濁液をPBSで1:10に希釈し、この希釈液の100μLを96ウェルプレートのウェルに移します。容器状の形態を呈するAD-MVFの全体数を計数し、全単離するために外挿されます。必要なAD-MVF濃度および精製は、個々に、インビトロまたはAD-MVF のin vivoアプリケーションでそれぞれに適合させることができます。これは、微小血管ネットワーク形成または組織欠損にインビボで移植するための足場上のAD-MVFの播種のインビトロ分析のためのコラーゲンゲルにおけるAD-MVFの埋め込みを含むことができます。 </li></ol>

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

本研究では、AD-MVFの単離のための十分に確立プロトコルを提示します。マウスの脂肪組織からの広告-MVFを取得することは、いくつかの重要なステップで簡単な手続きです。マウスは異なる皮下および腹腔内脂肪沈着を示します。以前にラットに関して記載したように、AD-MVFの単離のために最も適した脂肪源は、より大きな血管11との均質構造と最小限の汚染<sup class=...

組織工学は、 インビボの対応1,2 に動作不能の機能を回復または増強、維持、組織および器官代替の製造に焦点を当てています。設計組織構築物の運命は決定的に十分な血管新生3に依存します。これらの構成要素内の微小血管ネットワークが階層的に受信者の血管系4にinosculation後の効率的な血液灌流を可能にするために動脈、毛細血管、および細静脈で整理する必要があります。このようなネットワークの生成は、組織工学における主要な課題の一つです。この目的のために、実験的な血管新生戦略の幅広いスペクトルは、最後の二十年5、6の上に導入されました。 血管新生のアプローチは、設計TISSに受信者の微小血管の成長を刺激しますそのような成長因子7の取り込みなどの構造的または物理化学的骨格修飾によりUEの、。しかし、大規模な3次元構造の血管新生のために、血管新生依存戦略が著しく微小血管8の開発の遅い成長速度によって制限されています。 組織内の機能微小血管ネットワークの世代が前に彼らの移植の9に構築するためにこれとは対照的に、prevascularizationの概念が目指しています。従来prevascularizationは、足場内の内皮細胞、壁画細胞または幹細胞10などの血管形成細胞の共培養を含みます。微小血管ネットワークを形成した後、prevascularized構築物はその後、組織欠損に移植することができます。それは複雑で時間のかかるインビトロに基づいているため、注目すべきは、このprevascularizationアプローチは、臨床現場で適用することは困難です&lt;/ em&gt;の主要な規制のハードル9によって制限されている手順、。したがって、広範な臨床応用のために、より適している小説prevascularization戦略の開発の必要性が依然として存在しています。 そのようなprevascularization戦略は、脂肪組織由来の微小血管フラグメント（AD-MVF）のアプリケーションであってもよいです。 AD-MVFラット11、12及びマウス13の脂肪組織から大量に収穫することができる強力な血管新生単位を表します。彼らは細動脈、毛細管、及びルーメンと生理的な微小血管形態を示す細静脈血管セグメント、および安定血管周囲細胞14、15から成ります。このユニークな機能は、前培養することなく、組織欠損に広告-MVF-シードの足場の即時注入することができます。そこでは、広告-MVFは急速で組み立て直します機能的な微小血管ネットワークへ。また、AD-MVFは、さらにその打撃再生能力に寄与し得る間葉系幹細胞16の豊富な供給源を表します。したがって、AD-MVFはますます組織工学14、15、17、18、19、20、21の異なる分野で使用されています。 広告-MVFの単離は、もともとラット11、12に設立されました。ここで、我々は精巣上体脂肪パッドからマウス広告-MVFの標準化された単離を可能にするプロトコルを記述します。これは、トランスジェニックマウスモデルを使用して広告MVF機能の根底にある分子メカニズムさらなる洞察を提供することができます。

<table><tbody><tr><td>1.5-mL conical microcentrifuge tube</td><td>VWR, Kelsterbach, Germany</td><td>700-5239</td><td></td></tr><tr><td>100-&amp;micro;L precision pipette</td><td>Eppendorf, Hamburg, Germany</td><td>4920000059</td><td></td></tr><tr><td>10-mL measuring pipette</td><td>Costar, Corning Inc., New York, USA</td><td>4488</td><td></td></tr><tr><td>14-mL PP tubes</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>187261</td><td></td></tr><tr><td>1-mL precision pipette</td><td>Eppendorf, Hamburg, Germany</td><td>4920000083</td><td></td></tr><tr><td>500-&amp;micro;m filter (pluriStrainer 500 &amp;micro;m)</td><td>HISS Diagnostics, Freiburg, Germany</td><td>43-50500-03</td><td></td></tr><tr><td>50-mL conical centrifuge tube</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>227261</td><td></td></tr><tr><td>50-mL Erlenmeyer flask</td><td>VWR, Kelsterbach, Germany</td><td>214-0211</td><td></td></tr><tr><td>96-well plate</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>65518</td><td></td></tr><tr><td>cell detachment solution (Accutase)</td><td>eBioscience, San Diego, CA USA</td><td>00-4555-56</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River, Cologne, Germany</td><td>027</td><td></td></tr><tr><td>C57BL/6-Tg(CAG-EGFP)1Osb/J mice</td><td>The Jackson Laboratory, Bar Harbor, USA</td><td>003291</td><td></td></tr><tr><td>CD117-FITC</td><td>BD Biosciences, Heidelberg, Germany</td><td>553373</td><td></td></tr><tr><td>CD31-PE</td><td>BD Biosciences, Heidelberg, Germany</td><td>553354</td><td></td></tr><tr><td>Collagenase NB4G&amp;nbsp;</td><td>Serva Electrophoresis GmbH, Heidelberg, Germany</td><td>17465.02</td><td>Lot tested by manufacturer for enzymatic activity</td></tr><tr><td>Dissection scissors</td><td>Braun Aesculap AG &amp;amp;CoKG, Melsungen, Germany</td><td>BC 601</td><td></td></tr><tr><td>DNA-binding dye (Bisbenzimide H33342)</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>B2261</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle medium (DMEM)&amp;nbsp;</td><td>PAN Biotech, Rickenbach, Germany</td><td>P04-03600</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Biochrom GmbH, Berlin, Germany</td><td>S0615</td><td></td></tr><tr><td>Fine forceps</td><td>S&amp;amp;T AG, Neuhausen, Switzerland</td><td>FRS-15 RM-8</td><td></td></tr><tr><td>Fine scissors</td><td>World Precision Instrumets, Sarasota, FL, USA</td><td>503261</td><td></td></tr><tr><td>Dermal skin substitute (Integra)</td><td>Integra Life Sciences, Sain Priest, France</td><td>62021</td><td></td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Serumwerk Bernburg AG, Bernburg, Germany</td><td>7005294</td><td></td></tr><tr><td>M-IgG2akAL488&amp;nbsp;&amp;nbsp;</td><td>eBioscience, San Diego, CA USA</td><td>53-4724-80</td><td></td></tr><tr><td>Octeniderm (disinfecting solution)</td><td>Sch&amp;uuml;lke &amp;amp; Mayer, Norderstedt, Germany</td><td>118211</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Biochrom, Berlin, Germany</td><td>A2213</td><td></td></tr><tr><td>Petri dish</td><td>Greiner bio-one, Frickenhausen, Germany</td><td>664160</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Lonza Group, Basel, Switzerland</td><td>17-516F</td><td></td></tr><tr><td>pluriStrainer 20-&amp;micro;m (20 &amp;micro;m filter)</td><td>HISS Diagnostics, Freiburg, Germany</td><td>43-50020-03</td><td></td></tr><tr><td>Rat-IgG2akFITC</td><td>BD Biosciences, Heidelberg, Germany</td><td>553988</td><td></td></tr><tr><td>Rat-IgG2akPE</td><td>BD Biosciences, Heidelberg, Germany</td><td>553930</td><td></td></tr><tr><td>Small preparation scissors</td><td>S&amp;amp;T AG, Neuhausen, Switzerland</td><td>SDC-15 R-8S</td><td></td></tr><tr><td>Surgical forceps</td><td>Braun Aesculap AG &amp;amp;CoKG, Melsungen, Germany</td><td>BD510R</td><td></td></tr><tr><td>Tape (Heftpflaster Seide) 1.25 cm</td><td>Fink &amp;amp; Walter GmbH, Mechweiler, Germany</td><td>1671801</td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Bayer Vital GmbH, Leverkusen, Germany</td><td>1320422</td><td></td></tr><tr><td>&amp;alpha;-SMA-AL488</td><td>eBioscience, San Diego, CA USA</td><td>53-9760-82</td><td>Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)</td></tr></tbody></table>

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

機能的微小血管ネットワークは、改変された組織構築物の生存および統合のための極めて重要重要です。この目的のために、いくつかの血管新生およびprevascularization戦略が確立されています。しかし、ほとんどの細胞ベースのアプローチは、微小血管ネットワークの形成のために時間のかかるインビトロの工程を含みます。したがって、それらは手術ワンステップ手順には適していません。脂肪組織由来の微小血管フラグメント（AD-MVF）が有望な血管新生単位を表します。彼らは簡単に脂肪組織から単離され、機能的な微小血管の形態を示すことができます。また、彼らは急速にin vivoでの移植後の新しい微小血管のネットワークに組み立て直します。また、広告-MVFは、リンパ管新生を誘導することが示されています。最後に、彼らはさらに高い血管新生の可能性に寄与し得る間葉系幹細胞の豊富な源です。以前の研究では、我々は顕著vascularizatiを示しました設計骨や皮膚代替で広告-MVFの容量に。本研究では、我々は、マウスの脂肪組織からの広告-MVFの酵素を単離するための標準化されたプロトコルについて報告します。

本研究では、7〜12ヶ月齢のオスの野生型C57BL / 6マウス（35±1グラムの体重の平均）から、脂肪組織で6 AD-MVF単離手順を行いました。 図1は、後続の機械的および酵素AD-MVF分離有するマウス精巣上体脂肪パッドの採取を示す図です。脂肪の収穫に必要な時間は30分であり、AD-MVFの単離のために120分でした。合計では、手順が150分を要しました。 <p class="jove_c...

Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

我々は、有望な血管新生単位を表し、脂肪組織由来の微小血管断片を単離するためのプロトコルを提示します。それらはインビトロ処理に必要とせず、従って、組織工学の異なる分野でワンステップprevascularizationために使用することができる、迅速に分離することができます。

組織工学のための血管新生単位としてマウスの脂肪組織由来の微小血管フラグメントの単離

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

isolation-murine-adipose-tissue-derived-microvascular-fragments-as

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.4K ビュー.  Saarland University. 我々は、有望な血管新生単位を表し、脂肪組織由来の微小血管断片を単離するためのプロトコルを提示します。それらはインビトロ処理に必要とせず、従って、組織工学の異なる分野でワンステップprevascularizationために使用することができる、迅速に分離することができます。 

ビデオ: Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心筋組織工学のための生物由来注射用材料の作製

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

生物工学

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

組織工学のためのエレクトロ繊維のポスト処理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

微小血管断片からの血管新生熱発生脂肪組織の三次元培養

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

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

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

細胞表現型と分泌性特性化のためのヒト脂肪組織微細断片化

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

生物学

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

下流アプリケーションのための異なる脂肪デポからの血管内皮細胞の分離と同定(英語)

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

マウス白脂肪組織の半自動単離

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

著者スポットライト:組織解離器を使用したマウス白脂肪組織からの間質血管画分の半自動分離  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

マウス血管周囲脂肪組織から単離された前駆脂肪細胞の培養

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

著者スポットライト:マウス血管周囲脂肪組織からのSVF由来前脂肪細胞の単離、培養、および脂肪形成誘導の意義 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

組織工学のための血管新生単位としてマウスの脂肪組織由来の微小血管フラグメントの単離

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心筋組織工学のための生物由来注射用材料の作製

組織工学のためのエレクトロ繊維のポスト処理

微小血管断片からの血管新生熱発生脂肪組織の三次元培養

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

細胞表現型と分泌性特性化のためのヒト脂肪組織微細断片化

下流アプリケーションのための異なる脂肪デポからの血管内皮細胞の分離と同定(英語)

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

マウス大動脈周囲脂肪組織からの間質血管分画由来前駆脂肪細胞の単離・培養・脂肪誘発