Contreras and Watts Laboratoriesとウィリアム・ラファエル博士。これらの実験は、NHLBI F31 HL128035-01（組織消化プロトコル標準化）、NHLBI 5R01HL117847-02および2P01HL070687-11A1（動物）およびNHLBI 5R01HL117847-02（細胞単離および培養）によって支持された。

本書に記載されているすべての手順は、ミシガン州立大学の機関動物管理および使用委員会（IACUC）によって制定されたガイドラインに従います。すべてのバッファーとメディアを光から保護する必要があります。 緩衝液、培地および器具の調製<ol><li> （KRBB）：135mM塩化ナトリウム、5mM塩化カリウム、1mM硫酸マグネシウム、0.4mMリン酸二水素カリウム、5.5mMグルコース、1％抗生物質/抗真菌剤（10,000単位/ mLペニシリン、10,000μg / mLのストレプトマイシン、25μg/ mLのアンホテリシンB）、および10mMのHEPES（pH = 7.4）。この溶液は、4℃で滅菌した場合、3週間安定です。 </li><li>コラゲナーゼタイプ1溶液を調製する：4％ウシ血清アルブミン（BSA）を含むKRBB中に1mg / mL。この溶液は37℃で保存し、4時間安定です。 </li><li>赤血球溶解緩衝溶液の調製：154mM塩化アンモニウム、10mM重炭酸カリウム、および0.1mMのEDTAを含む。 4℃で1ヶ月間保管してください。 </li><li> DMEM / F12培地、10％ウシ胎仔血清（FBS）、5％正常ロバ血清、40μL/ mL F（ab）断片ロバ抗ラットIgGを調製する。 4℃で1ヶ月間保管してください。 </li><li> MCS緩衝溶液：リン酸緩衝食塩水（PBS、pH = 7.5）、0.5％BSA、および2mM EDTAを調製する。 4℃で1ヶ月間保管してください。ガラス容器内で37℃に加熱し、次いで15秒間真空を適用することによって、脱ガス溶液。使用しないでください。 </li><li> （DMEM）：F12,15％ウシ胎児血清（FCS）、1％抗生物質/抗真菌剤（10,000単位/ mLペニシリン、10,000μg/ mLストレプトマイシン、25μgのストレプトマイシン、 / mLアンホテリシンB）、44.05mM重炭酸ナトリウム、100μMアスコルビン酸、33μMビオチン、17μMパントテン酸塩、2mM L-グルタミン、および20mM HEPES。無菌状態で4℃で2週間まで保存してください。 </li><li> APC＆白血病抑制因子（10 ng / mL）、血小板由来増殖因子BB（10 ng / mL）、および塩基性線維芽細胞増殖因子を含む基礎増殖培地（＃160;培地： 5ng / mL）。無菌状態で4℃で2週間まで保存してください。 </li><li> APC誘導培地：10％FBS、2.5μg/ mLインスリン、0.5mM 2-イソブチル-1-メチルキサンチン（IBMX）、1μMデキサメタゾン、および200μMT3（トリヨードチロニン甲状腺ホルモン）を含むAPC培地を調製する。無菌状態で4℃で2週間まで保存してください。 </li></ol> 2.脂肪細胞前駆細胞の単離<ol><li>施設ガイドラインに従ってラットを麻酔する。ラットを背臥位に置く。つま先のつまみで麻酔の深さを確認し、この痛みを伴う刺激に対する反射応答の喪失を確認します。 注：このプロトコールは、10週齢のSprague Dawleyラットおよび70mg / kgのペントバルビタールを腹腔内注射によって送達する。 </li><li> 垂直正中線切開を作る胸骨領域の胸骨に沿って、および会陰領域にi番目のはさみ。腹腔に入り、上腸間膜動脈、小腸腸間膜抵抗性血管（mPVAT）および胸部大動脈（aPVAT）を暴露する。 <ol><li>腸間膜および大動脈への接続をすべて切断し、動物から血管を取り除く。解剖顕微鏡とKRBBで満たされたペトリ皿を用いてPVATを分離し、血管を観察し、PVATを単離する。 </li><li>この実験では、非PVAT脂肪蓄積を表す生殖腺（GON）脂肪を収集する。 10mMのHEPES（pH = 7.4）を含むKRBBの氷上に単離された脂肪パッドを置く。 </li><li>バイオセーフティフードでは、約50mgの組織を、1mLのコラゲナーゼI型溶液を含む1.7mLチューブに移し、組織はさみ（1〜3mm片）で切ります。 </li></ol></li><li> Rotisserieインキュベーター（またはオービタルシェーカーを備えたインキュベーター）で37℃で1時間インキュベートしてサンプルを消化する。バイオセーフティフードでは、消化した物質を100および40<html> 50mLのチューブに移した。得られたろ液を4℃で10分間、300×gで遠心分離する。 注記：ここからのプロトコールの全てのステップは、細胞を無菌状態に保つためにバイオセーフティフード内で実施するものとする。 </li><li>上清を除去し、SVF細胞を含むペレットを1×赤血球溶解緩衝溶液1mLに再懸濁し、1.7mLの微量遠心管に移す。光から保護されたRTで5分間細胞をインキュベートし、4℃で300xgで5分間遠心分離する。 </li><li> SVF Basal Mediaに上清を残し、残りの細胞ペレットを再懸濁する。トリパンブルー溶液を用いて生細胞を計数するために20μLのサブサンプルを収集する。 注：隔離可能なSVF細胞の数は、サイトによって異なります。組織1mg当たりに採取されたSVFの平均数は、aPVAT = 5.0±2.0×10 3 、mPVAT = 1.04±0.62×10 4 、GON = 2.4±1.2× 10 5である 。 </li></ol> 3.磁気活性セルソーティング_content &quot;&gt;注：4℃ですべてのステップを実行することにより、CD34およびPDGFRα細胞表面マーカーに基づいてSVFからAPCを単離します。 <ol><li>細胞を300 x gで5分間スピンします。上清を捨て、細胞ペレットを1×10 6細胞/ mLでMCSブロッキング緩衝液に再懸濁し、20分間インキュベートする。 注：1×10 6〜2×10 8細胞/ mLの細胞懸濁液を効果的に分離することができます。 </li><li> 細胞を5μLのFITC結合マウス抗CD34（1μg/ 1×10 6細胞）と共に4℃で30分間インキュベートする。 300 xgおよび4℃で5分間細胞をスピンします。 <ol><li> 4μLの抗FITCマイクロビーズと96μLのMCSバッファー（全量100μL）と4℃で5分間、暗所でインキュベートし、CD34 +およびCD34 -細胞を分離する。 </li></ol></li><li>磁気分離器をスタンドに取り付け、列翼を前面に向けてMultiSort（MS）Columnをセパレータに置きます。 5 mL上部チューブホルダー内のMSカラムの下に置いてください。 </li><li> MCS Buffer SolutionでリンスしてMSカラムを調製します。カラムの上に500μLのMCS Bufferをアプライし、バッファを通過させます。流出液を廃棄し、回収チューブを交換する。 </li><li>抗体標識された細胞懸濁液を調製したMSカラムにロードする。未標識細胞を含むフロースルーを収集する。 </li><li>脱気したMCS緩衝液3×500μLでMSカラムを洗浄する。前のステップからの流れを通過して結合する非標識細胞を収集する。 </li><li>磁気分離器からMSカラムを取り外し、新しいコレクションチューブ上に置きます。 1mLのMCS緩衝液をMSカラムに注ぎます。直ちに磁気標識した細胞でフラクションをただちに洗い流しますが、カラムに供給されたプランジャーをゆっくりと加えて、余分なガスがカラムに入らないようにしてください。 注：単離された細胞数はサイトによって異なります。 SVF集団から単離されたCD34 + APCの平均数組織のmgは、aPVAT = 2.6±0.43x10 2 、mPVAT = 9.6±1.4x10 2 、GON = 1.3±0.22x10 3である 。 </li><li> 細胞を300×gおよび4℃で5分間スピンさせる。 1：200溶液/ 1×10 6細胞ウサギ抗PDGFRα10μL中に集めたCD34 +画分を4℃で30分間インキュベートする。 <ol><li> 300 xgおよび4℃で5分間細胞を再度遠心する。標識された細胞を抗ウサギIgGマイクロビーズ4μLおよびMCS緩衝液96μLとインキュベートし、3.3から3.7のステップを繰り返して単離する。 注：単離された細胞数はサイトによって異なります。組織1mgあたりのCD34 +集団から単離されたPDGFRα + APCの平均数は、aPVAT = 2.4±0.64、mPVAT = 8.4±2.4、GON = 10.4±1.9であり、これは以前に単離された集団の0.5~10％である。 </li></ol></li></ol> 4.細胞培養および脂肪生成誘導<ol><li> SVFの文化および2日間毎に交換する基礎培地中の6ウェル組織培養プレート中のAPCを含む。 3回の連続継代後、8ウェルプレート、24ウェルプレート、48ウェルプレート、96ウェルプレート、24ウェルプレートで50,000細胞/ウェルで評価される増殖アッセイのために1×10 2細胞/ウェルの黒色96ウェル組織培養プレートにプレートするか、定性的および定量的である脂肪生成アッセイのための48ウェル組織培養プレート中の10,000細胞/ウェル。 </li><li>分化のために示されたように、骨形成タンパク質4（3.3nmol / L）によるコンフルエント後48時間および誘導前のAPC基礎培地の補充。細胞をインキュベートするためにAPC誘導培地を用いて100％コンフルエンシー（0日目）の48時間後に細胞を誘導する。 </li><li> 48時間後、培地を交換して、IBMXおよびデキサメタゾンを含まないAPC誘導培地中で、14時間毎に培地を交換し、48時間ごとに細胞を維持する。 </li><li> FACSによって検証されたMCS分離。 <ol><li>磁気的に分離した50μL（50,000セル）のサブサンプルを取るFACS溶液で洗浄する。 </li><li>細胞を遠心分離し、PDGFRα +細胞を標識するロバ抗ウサギIgG Dylight 405の1：1,000溶液100μL中に再懸濁する。軽く、4℃で30分間インキュベートする。 </li><li>フローサイトメーターで488 nm（FITC）および405 nm（Dylight 405）フィルターを使用して解析するまで、細胞を洗浄し、2％ホルムアルデヒド溶液200μLで再懸濁します。 注：培養細胞による脂質蓄積は、蛍光を測定するマイクロプレートリーダーで脂質親和性脂肪生成蛍光アッセイを使用し、脂質蓄積のキャリブレーターとして前脂肪細胞を使用して定量的に評価する。脂質蓄積はまた、脂質色素を染色するかまたは含まない全細胞のパーセンテージを観察可能にする、カメラを備えた倒立顕微鏡で行われる脂質色素染色およびイメージングによって定性的に測定される。 485nmでの励起および572nmでの発光で蛍光を測定することができる任意のプレートリーダーnmは、デジタル画像を取り込むことができるカメラを備えた顕微鏡と同様に、分析にも適しています。 </li></ol></li></ol>

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F., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+systems+and+their+applications+in+drug+discovery+and+cell-based+biosensors.">Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors.</a> Assay Drug Dev Technol. 12 (4), 207-218 (2014).</li></ol>

著者は何も開示することはない。

本実験の中心的な焦点は、PVATデポからのAPCの単離、拡大、および脂肪生成誘発である。ここでは、表面マーカーCD34およびPDGFRαを発現する細胞の同定に基づいて、APCの単離のためのプロトコールを提示する。これらの表面タンパク質は、APC上で、高い増殖速度および様々な脂肪貯蔵庫14,15中の白色または褐色脂肪細胞に分化する可能性を伴って以前に同定された。これらの?...

脈管周囲脂肪組織（PVAT）は、血管に近接しているため、脈管機能1における主要なパラクリンシグナル伝達成分である。この脂肪組織の拡大は、存在する脂肪細胞前駆細胞（APC）の表現型に依存する2,3 。脂肪組織からの細胞の単離は、主要な脂肪細胞が壊れやすく、浮力があり、大きさの範囲があるため、困難である。特定の単離技術はまた、炎症性タンパク質合成を増加させ、脂肪生成遺伝子発現を減少させることによって細胞表現型および形態を変えることができ、細胞の完全性を維持するプロトコルの重要性を強調する。 初代細胞および特異的前脂肪細胞亜集団の培養は、インビボ増殖に対する還元主義的アプローチを提供し、同等の細胞遺伝的メイクアップ5を維持するが、これらの細胞の私は、老化や老化による劣化のために制限されています6 。皮下および大網デポーを含む様々な脂肪デポーからの前脂肪細胞もまた、特定の解剖学的部位から細胞を集めることの重要性を強調する増殖の差異7を示す。非PVAT白色脂肪細胞由来の前駆細胞は、以前の研究7,8,9において特徴付けられてきたが、PVAT APC表現型についてはあまり知られていない。 ここに記載された技術は、形態学、生存能力、および増殖および分化の可能性に最小限の影響を及ぼしながら、特異的および限定されたAPC集団の分析を可能にする。ビーズが細胞を変えずに溶解するので、磁気活性化細胞選別（MCS）は、培養などの下流の適用に適している。 MCSもまた経済的であり、一旦、抗体の欠点フローサイトメトリーアッセイの必要性は最小限に抑えられています。 PVAT前駆体を用いたインビトロ研究はまた、これらの初代細胞が有する可能性を垣間見ることができる。

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:325px;">Tissue Dissection</td>
			<td style="width:168px;"></td>
			<td style="width:108px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 170</td>
			<td>Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splinter Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>105-400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KRBB Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7758-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic/Antimicotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type 1</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2876</td>
			<td>Reciprocal Shaking Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Cabinet</td>
			<td>Thermo-Fisher Scientific</td>
			<td>1385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotisserie Incubator</td>
			<td>Daigger</td>
			<td>EF4894C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-549</td>
			<td>Yellow</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Blue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OctoMACS Kit</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(DMEM):F12 Medium</td>
			<td>Corning</td>
			<td>90-090</td>
			<td>Medium Base</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35016CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Donkey Serum</td>
			<td>AbCam</td>
			<td>AB7475</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD34</td>
			<td>Santa Cruz</td>
			<td>SC-7324</td>
			<td>FITC conjugated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PDGFR&#945;</td>
			<td>Thermo-Fisher Scientific</td>
			<td>PA5-17623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-007-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS 10X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 Cell Incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>51030285</td>
			<td>Heracell 160i&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48- Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Plates, Black Wall</td>
			<td>Corning</td>
			<td>353376</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Calf Serum</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>137-08-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Corning</td>
			<td>61-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone Morphogenic Protein 4</td>
			<td>Prospec Bio</td>
			<td>CYT-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal Growth Factor</td>
			<td>PeproTech</td>
			<td>400-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia Inhibitory Factor</td>
			<td>PeproTech</td>
			<td>250-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived Growth Factor BB</td>
			<td>Prospec Bio</td>
			<td>CYT-740</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Corning</td>
			<td>25-800-CR</td>
			<td>ITS Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>28822-58-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>50-02-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T3 (Triiodothyronine)</td>
			<td>Sigma-Aldrich</td>
			<td>6893-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyQUANT Proliferation Assay</td>
			<td>Thermo-Fisher Scientific</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AdipoRed Fluorescence Assay Reagent</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O Lipid Dye Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>O1391</td>
			<td>In Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M1000 Microplate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Sight DS-Qil Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

パラクラインシグナリングを介した血管機能の主要調節因子である血管周囲脂肪組織（PVAT）の拡大は、肥満時の高血圧の発症に直接関連する。肥大および過形成の程度は、デポの位置、性別、および存在する脂肪細胞前駆細胞（APC）表現型のタイプに依存する。過去10年間のAPCおよび前脂肪細胞分離のために使用された技術は、個々の細胞が特定の細胞表面マーカーに基づいて同定され得る正確さを大幅に改善した。しかしながら、APCおよび脂肪細胞の単離は、特にインタクトな細胞を細胞培養適用のために保持しなければならない場合、細胞の脆弱性のために困難であり得る。 磁気活性化細胞選別（ MCS）は、脂肪組織の重量単位あたりより多くの生存APCを単離する方法を提供する。 MCSによって回収されたAPCは、インビトロプロトコールでプレア成長因子カクテルを使用してそれらを脂肪細胞に分化させ、細胞によって保持された多分性および脂肪生成能の分析を可能にする。この実験は、拡張中の心臓血管疾患の発症において重要な役割を果たす大動脈および腸間膜のPVATデポーに焦点を当てた。これらのプロトコルは、定義されたAPC集団を分離し、拡大し、そして区別するための方法を記載する。このMCSプロトコルは、最小限の設備またはトレーニングで細胞選別が必要なあらゆる実験で分離を使用することを可能にする。これらの技術は、細胞表面マーカーの存在に基づいて特定の細胞集団の機能性を決定するさらなる実験を助けることができる。

前脂肪細胞の増殖能力および脂肪細胞前駆体の脂肪生成能は、インビトロで維持される特性である11 。定量的DNAアッセイを用いてプレーティングした8,24,48および96時間後に、雄ラットのaPVAT、mPVAT、およびGONから単離したSVFおよびAPCのin vitro増殖を評価した。同じサイトのSVF細胞（ 図1 ）と比較して96時間の増殖が?...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

ここでは、MCS（Magnetic-Activated Cell Sorting）を用いた脈管内脂肪組織（PVAT）からの脂肪細胞前駆細胞（APC）集団の単離方法を報告する。この方法は、蛍光活性化細胞選別（FACS）と比較した場合、脂肪組織のグラム当たりのAPCの単離の増加を可能にする。

磁気活性化細胞選別法により単離された血管周囲脂肪組織からの脂肪細胞前駆体の拡大および脂肪生成誘導

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

expansion-adipogenesis-induction-adipocyte-progenitors-from

Research

JoVE Journal

Developmental Biology

9.4K ビュー.  Michigan State University. ここでは、MCS（Magnetic-Activated Cell Sorting）を用いた脈管内脂肪組織（PVAT）からの脂肪細胞前駆細胞（APC）集団の単離方法を報告する。この方法は、蛍光活性化細胞選別（FACS）と比較した場合、脂肪組織のグラム当たりのAPCの単離の増加を可能にする。 

ビデオ: Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

発生生物学

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.

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.

ブタ皮下脂肪組織から分離し、脂肪由来幹細胞の分化

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

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

単離およびヒト胎盤組織由来の間葉系幹/間質細胞の増殖

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

ヒト心臓組織からの血管周囲多能前駆細胞集団の単離

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

心臓毒注入による急性骨格筋再生の誘導

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

遠心重力によって脂肪由来幹細胞の軟骨形成分化誘導

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

フローサイトメトリーによるマウス胚における赤血球系前駆細胞およびその子孫の同定

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

精巣上体から分離した一次上皮細胞の全細胞パッチクランプ記録

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

マウス膵臓前駆細胞の in Vitroからスキャフォールド フリー、三次元のインスリンを表現する Pancreatoids の生成

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.