私たちは社説助けをジェニファーPetrosinoのとDavid DiSilvestroに感謝したいと思います。本研究では、アメリカの卵会およびノボノルディスクファーマから賞番号10040042からだけでなく、OSUでの食品イノベーションセンター、オフィス国際のための、高度な機能性食品研究センター、および起業家精神によってだけでなく、受賞番号20020728でサポートされていました全米科学財団は、EEC-0914790（LJL）を付与します。このプロジェクトは、ディレクターのオフィスによって資金を供給、研究資源のための国立センターから賞数R21OD017244（OZ）とUL1RR025755（OSUCCC）でサポートされている国立衛生研究所（OD）と医学研究のためのNIHロードマップによってサポートされていました説明とNCI P30CA16058。内容はもっぱら著者の責任であり、必ずしも研究資源のための国立センターや国立衛生研究所の公式見解を示すものではありません。

研究プロトコルは、オハイオ州立大学の倫理委員会によって承認されました。動物実験は、IACUCプロトコルによって承認されました。すべての手順は、層流とレベル2生物学的安全キャビネットの下で行いました。我々は、すべての標準的な安全要件と手順に従いました。 17、18に記載されているように、マイクロカプセルの製造のためのマイクロカプセル化技術が行われています。 材料の1の準備<ol><li>オートクレーブ処理生理食塩水（水中の0.9％NaCl）で2％アルギン酸ナトリウム溶液10mlを準備します。生理食塩水中の0.05％PLL溶液を調製します。前日これらのソリューションを用意し、一晩攪拌します。使用前に0.22μmフィルターでソリューションをフィルタリングします。 </li><li> 0.9％NaCl溶液に50 mMのクエン酸ナトリウム及び100mMのCaCl 2を用意し、それらをオートクレーブ。 </li><li>すべてのソリューション、針、電極、およびビーカーをオートクレーブ。目詰まりを回避するために、ワイヤで十分にきれいな針。 </li><li> （102マイクロカプセルがよく毎cm 2のために必要とされており、カプセル18あたり約500個の細胞がある）カプセルに使用する細胞の適切な量を決定します。 200万細胞あたりアルギン酸ナトリウムの1ミリリットルを使用してください。 </li><li> 「線維芽細胞増殖培地は、「10％ウシ血清を含む用意し、高グルコースで100 U / mlペニシリン/ストレプトマイシン（4,500 mg / Lのグルコース）ダルベッコ改変イーグル培地（DMEM）。 <ol><li> DMEM、10％ウシ胎児血清、10μg/ mlインスリン、1μMのデキサメタゾン、0.5mMの3-イソブチル-1-メチルキサンチン、および100 U / mlのペニシリン/ストレプトマイシンを含有する「分化培地I &#39;を準備します。 </li><li> DMEM中で「分化培地II &#39;を含む10％ウシ胎児血清、10μg/ mlのインスリン、および100 U / mlペニシリン/ストレプトマイシンを準備します。 </li></ol></li><li> 10ミリリットルラジオ免疫沈降アッセイバッファごとにプロテアーゼ阻害剤錠剤を含む溶解緩衝液を調製する（RIPA緩衝液）。 </li></ol> 2.アルギン酸マイクロビーズの調製（図1A） <ol><li>細胞培養フラスコから古い培地を除去します。 10mlのPBSで細胞を洗浄します。 0.25％トリプシン-EDTA（1コンフルエントのT175フラスコ当たり2ml）で懸濁液中の細胞を持参してください。 </li><li>血球計数器を用いて細胞懸濁液中の細胞をカウントします。細胞懸濁液から10ミリリットルのアリコートを使用し、製造業者の説明書に従って細胞をカウントします。室温で5分間480×gの遠心分離培地中に残存する細胞を遠心します。 </li><li>ステップ1.4で説明したようにアルギン酸ナトリウムで細胞ペレットを一時停止します。 5ミリリットル注射器にアルギン酸ナトリウム - 細胞溶液を移します。 </li><li> 23ゲージの針を追加し、溶液中の気泡を除去し、空気1mlのポケットを作成するための注射器を反転。 </li><li>カプセル化の針注ぎ口の下の100mMのCaCl 2溶液144ミリリットルを含む小さなビーカー（180ミリリットル）を配置します。先端approximでカプセル化に電極を取り付けately 2.5センチメートルの100mMのCaCl 2溶液の表面の上。 </li><li>しっかりとシリンジポンプでのアルギン酸ナトリウム - 細胞溶液を含む注射器を置きます。注射器の開口部にゴムチューブを取り付けます。アルギン酸ナトリウム - 細胞溶液がチューブの途中に入るまで、プランジャーを押してください。 5.4 kVのに電圧を調整します。シリンジポンプの12.06ミリメートルの直径を設定します。 3ミリリットル/時の速度を調整します。ポンプを起動します。カプセル化をオンにして、5.4 kVの電圧を維持します。 </li><li>フードのウィンドウを閉じ、アルギン酸マイクロビーズの形成が終了するまで、不要な振動を避けることができます。すべてのソリューションは、針を通過した後、PLLでコーティングする前に、追加の20分間の100mMのCaCl 2溶液中のアルギン酸ボール状のマイクロビーズを固めます。 </li></ol> PLL 3.コーティングマイクロビーズ（図1B） <ol><li>アルギン酸ナトリウムセルボール状のマイクロビーズを含むビーカーを削除し、これらがMicroBを転送50ミリリットルの遠心チューブにEADS。マイクロビーズペレットからのCaCl 2溶液を除去します。 </li><li> 0.9％NaClを30mlのを追加することにより、アルギン酸セルボール状のマイクロビーズを洗ってください。そっと手でチューブを振ります。マイクロビーズは、重力によって沈殿させた後、25ミリリットルピペットで0.9％のNaClを削除します。合計3回の洗浄のためにさらに2回繰り返します。 </li><li>アルギン酸ナトリウム溶液の各1ミリリットルのための0.05％PLL溶液10mlを使用してください。 10分間分1,000回転で0.05％のPLLと渦を追加します。 注意：通常、10分PLLコーティングのために十分です。 </li><li> PLLコートを形成した後、PLL溶液を除去し、3.2に記載したように、カプセルを3回洗浄します。 </li></ol>アルギン酸コアの4除去（図1C） <ol><li> 50mMのクエン酸ナトリウム溶液30mlを加えます。 5分またはすべてのアルギン酸ナトリウムが溶解するまで待ってください。ステップ3.1.1で説明したように洗浄を3回カプセル。 </li><li> 0.9％のNaClを削除し、50mlチューブに培養液の20ミリリットルを追加します細胞培養フラスコに細胞を含むすべてのカプセルを転送します。標準的な細胞培養条件18歳未満のカプセル化された細胞を処理します。 </li></ol>異種移植と細胞間の代謝物の流入/流出の宿主細胞間相互作用やキネティクス（図2）を研究するためのインビトロ適用5. <ol><li>共培養のためにコンフルエントになるまで24ウェルプレート上で培養された宿主細胞。脂肪前駆細胞を培養するため「線維芽細胞増殖培地 &#39;を使用します。 </li><li>コンフルエント宿主細胞を含む24ウェルプレートにマイクロカプセルを転送します。単層（ウェルの102マイクロカプセル/ cm 2の ） を達成するために、マイクロカプセルを追加します。 </li><li> 6日間分化培地IIへの前脂肪分化培地Iと分化48時間ごとに、変化媒体を誘導します。 RIPA緩衝液中で細胞を溶解します。ウエスタンブロットを用いて、タンパク質の発現を分析するために条件当たり50μgのタンパク質を使用してください。 </li></ol> Treatmためのインビボ応用6.肥満のENT（図3） <ol><li>麻酔の麻酔の導入および維持のための酸素と2％のイソフルランのための酸素と4％のイソフルランを混ぜます。つま先のピンチにより十分な麻酔深度を確認してください。 <ol><li>乾燥から角膜を保護するために、目のかゆみ止め軟膏を適用します。 </li></ol></li><li>このような緑色蛍光タンパク質（GFP）のような恒久的に人工蛍光タンパク質で標識されたカプセル化細胞を使用します。 </li><li>カプセル化された細胞を注入するために3ミリリットルの注射器と20ゲージの針を使用してください。 <ol><li> 図3に示すように、生殖腺と腎臓の間腹腔内領域に配置されている各IAB脂肪デポに0.2mlのPBSに懸濁し、0.5×10 6個の細胞を注入します。の平均重量を有するマウスは、このPBSの容量とセル数を使用します40g添加しました。異なる重量または用量依存効果の決意のために持っているマウスでは細胞数と音量を調整します。 </li></ol></li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meteorin-like+is+a+hormone+that+regulates+immune-adipose+interactions+to+increase+beige+fat+thermogenesis.">Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis.</a> Cell. 157, 1279-1291 (2014).</li><li>Kir, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-derived+PTH-related+protein+triggers+adipose+tissue+browning+and+cancer+cachexia.">Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia.</a> Nature. 513, 100-104 (2014).</li></ol>

No conflicts of interest declared.

種々の方法は、乾燥、押出し、および乳剤19を含む細胞をカプセル化するために使用されてきました。この方法では、アルギネートビーズをPLLでコーティングし、アルギン酸塩コアをカプセル化を完了するために溶解され、針を通して押し出されます。この方法は、長年使用されているが、所望のサイズおよび形状の球形ビーズの形成は依然として困難です。カプセルのサイズは、ア...

慢性疾患の発生増加1は、治療用細胞集団2の移植に関する研究を刺激してきました。同系または同種異系幹細胞は、これらのアプリケーション2のために最も一般的に使用される細胞型です。しかし、これらの治療は、移植後の幹細胞の分化と移動の制御を可能にしないと、効率的なコストされていません。有益な機能を有する遺伝的に改変された細胞の移植は多くの疾患の治療を改善すると予想しています。しかし、遺伝子の細胞改変は宿主の免疫系によって認識され、したがって、これらの治療は、免疫抑制3が必要です。インスリン産生細胞のカプセル化は、チャン博士4によって開発されました。技術は、塩化カルシウム溶液中に浸漬されたアルギン酸塩の液滴中の細胞のカプセル化に基づいています。アルギン酸分子は、（M）マンヌロンとグルロン酸（G）で ​​構成され、CAによって接続することができ2+。ゲル化後、ビーズを、ポリ-L-リジン（PLL）溶液に懸濁させます。このステップの間、PLLは、カプセルの膜を確立アルギン酸分子中のGとMに結合します。カプセルの膜の多孔度は、MとPLLの濃度、インキュベーション時間、温度を変化させることによって調節することができます。 PLLの結合はまた、アルギン酸塩の種類と濃度に依存します。のCa 2+イオンで ​​架橋されたアルギン酸塩マトリックスは、生理的環境中またはリン酸とクエン酸イオン濃度の高い共通の緩衝溶液中で不安定です。これらのバッファは、アルギン酸からのCa 2+を抽出し、コアを液化することができます。アルギン酸塩コアの液状化は、細胞の移動および増殖のためのカプセルの内部の空間を提供します。ポリカチオン性ポリ-L-リジン（APL）とポリアニオンのアルギン酸に封入された細胞は、免疫グロブリンのために不浸透性であるが、毒素の栄養素の流入と流出を持っています。これらのAPLの特性は、長期のsuを有効に遺伝的に異なるホストへの移植後にカプセル化された細胞のrvival。エリオットらは、9年後の移植5人の患者にカプセル化されたブタの膵臓細胞を機能の生存を報告しました。 カプセル化技術は、マイクロカプセル化（3-800ミクロン）およびマクロカプセル（千ミクロンよりも大きい）に分類することができます。マイクロカプセルは、マクロカプセル6よりも耐久性があります。 1964年のチャン博士らによるその発見以来、マイクロカプセル化は広く、インスリン、他のホルモン、および生理活性分子7を製造同化細胞のカプセル化のために使用されてきました。これらの治療は、線維症、免疫応答8を含む宿主組織内のいくつかの課題に直面していました。最初は、バイオポリマーの品質に関連する副作用が解決されました。しかし、同化細胞の移植は、依然としてホルモンの結果として、そのような線維症などの副作用を開始overpr特殊な腺の外側oduction。 ここ数十年では、肥満や2型糖尿病は、大流行9に達しています。大人の人の30％以上は、世界中の過体重および肥満である10。増加腹腔内（IAB）脂肪形成は、慢性炎症の発生率を増加させ、2型糖尿病、心血管疾患、特定の癌および他の病的状態11-13を促進します。いくつかの証拠は、IABの脂肪に関連する病因が特定の脂肪細胞によって回避することができることを示唆しました。最近の研究では、IAB領域への皮下の脂肪細胞の移植は、代謝を改善し、インビボで 14げっ歯類の肥満およびインスリン抵抗性を減少させることができることを示しました。肥満とインスリン抵抗性の効果的な低減は、熱15,16の形でエネルギーを放散することが可能な熱発生、脂肪細胞と関連しています。脂肪細胞の産熱変性は、安定なトランスフェクションによって達成することができますこのような脱共役タンパク質1（UCP1）またはUCP1および他サーモ15,16遺伝子の発現を調節する遺伝子のミトコンドリアのプロトン脱共役に関与する遺伝子の。我々の最近の研究では、アルデヒドデヒドロゲナーゼの欠乏1 A1（ALDH1A1）が 17,18、これらのマウスにおいて、肥満およびインスリン抵抗性を減少させるIAB脂肪の熱発生リモデリングをもたらすことを示しました。なお、熱発生ALDH1A1欠損（ALDH1A1 - / - ）のカプセル化前脂肪細胞は、IAB脂肪18の治療のための新規治療機会を示唆し、肥満の野生型マウスでIAB脂肪の同じ治療効果を媒介します。実験的な設定では、カプセル化された細胞は、費用対効果の高い方法19内の特定の細胞集団の効果を研究するために研究者を可能にします。ここでは、熱発生異化細胞株とその研究室のカプセル化と肥満のマウスモデルにおける治療的適用の方法について説明します。プロトコルは、Tを説明しますマイクロカプセルの製造（ 図1）のためのHREE相：アルギン酸ビーズ（ 図1A）、ポリカチオン性ポリ-L-リジン（PLL）マイクロビーズの表面（ 図1B）上の膜の形成および除去の形成アルギン酸コア（ 図1C）。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Encapsulation device (VAR V1)</td>
			<td>Nisco</td>
			<td>LIN-0042</td>
			<td>None</td>
		</tr>
		<tr>
			<td>KD scientific syringe pump</td>
			<td>KD scientific</td>
			<td>780100Y</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Olympus microscope&#160;</td>
			<td>Olympus Optical</td>
			<td>IX70-S8F2</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma</td>
			<td>MKBP8122V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Poly-l-lysine hydrobromide (PLL)</td>
			<td>Sigma</td>
			<td>020M5006V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>SLBJ2662V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma</td>
			<td>030M0200</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>SLBD2595V</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Gels</td>
			<td>Bio-Rad</td>
			<td>456-1093</td>
			<td>None</td>
		</tr>
		<tr>
			<td>ATGL primary antibody (from rabbit)</td>
			<td>Cell Signaling</td>
			<td>2138S</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Secondary anti body (anti rabbit)</td>
			<td>LI-COR</td>
			<td>926-68071</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Radio-Immunoprecipitation Assay (RIPA) buffer</td>
			<td>Boston BioProducts</td>
			<td>D25Y6Z</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>RNBD2893</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Cortizone 10 anti-itch ointment</td>
			<td>Cortizone 10</td>
			<td>C4029138</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Newborn calf serum (CS)</td>
			<td>Sigma</td>
			<td>N4762</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>None</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Insulin (bovine)</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

細胞カプセル化は、半透過性膜内に生存細胞を捕捉するために開発されました。移植カプセル化細胞は、長期生存を達成するために、処置される宿主の組織中に低分子量の代謝産物を交換することができます。半透膜は、移植されたカプセル化された細胞は、免疫系による拒絶反応を回避することができます。カプセル化手順は、例えば、インスリン、他のホルモン、およびサイトカインなどの生物活性化合物の制御放出を可能にするように設計されました。ここでは、肥満マウスの腹腔内脂肪組織における熱産生及びエネルギー散逸のための脂質を消費異化細胞、（熱発生）のカプセル化のための方法を説明します。サーモ異化細胞のカプセル化は、肥満の予防と治療に潜在的に適用可能であること、および2型糖尿病があります。異化細胞の別の潜在的なアプリケーションは、アルコールまたは他の毒性代謝物および環境汚染物質の解毒を含むことができます。

図1は、マイクロビーズの生産の各ステップは、顕微鏡下で制御することができたことを示している。 図2（a）は、共培養する方法脂肪細胞をカプセル化された細胞の単層で示している。 図2Bは、脂肪細胞/マイクロカプセルの共培養を用いた定量的研究の代表例であること脂肪細胞の前記溶解物を、ウェスタンブロットを用いて分析したセクションで説?...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

移植のためのカプセル化サーモ前脂肪細胞の脂肪組織デポへ

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

9.2K ビュー.  The Ohio State University. Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

ビデオ: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACSの精製された感光体前駆体細胞の成人マウス・レチナへの副腎移植

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

医学

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

異心臓移植のマウスモデルを用いた肺動脈弁の移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

間褐色脂肪組織のチェレンコフ発光イメージング

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

マウス脂肪デポのローカライズ、識別、および切除

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

人間の褐色脂肪組織デポは自動的に陽電子放射断層撮影/コンピュータ断層撮影法で区分されると磁気共鳴画像登録

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

マウスの卵巣脂肪パッドに移植

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

非ジアテルミー法による腹部皮下脂肪の生検のためのテクニック

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

顕微鏡を用いたひと脂肪組織血管機能評価

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

ラットモデルにおける部分的な術後後の漏れを減らすために脂肪組織由来幹細胞シートの移植

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

潜在的な軟骨再生のための脂肪組織由来間質血管分数の生産の臨床プロトコル

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...