我々は、FACS選別の支援のためのメイヨークリニック顕微鏡細胞分析コアフローサイトメトリー施設を認める。

すべての動物実験手順は、メイヨークリニックの施設動物ケアおよび使用委員会の承認を得て行われました。
1. ソリューションの準備 
<ol><li>コラゲナーゼII 2 gを100 mLハンクスバランス塩溶液(HBSS)に溶解して2%(w/v)溶液を調製します。アリコート 200 μL 各用途に適しています。</li>
	<li>84 mL F-12培地、15 mL馬血清、および1 mLペニシリン/ストレプトマイシンを混合して中和培地を調製します。</li>
	<li>500 mgのウシ血清アルブミン(BSA)と400 μLの0.5 M EDTAをダルベックコのリン酸緩衝生理食塩基(DPBS)の100 mLに溶解してフローサイトメトリーバッファーを調製します。</li>
</ol>2. 上端体脂肪パッドの解離と組織解離
<ol><li>人道的に安楽死させる(イオブルラン/子宮頸部脱臼)1つの雄マウス。このプロトコルでは、4か月のFVBマウスが使用されました。</li>
	<li>腹部に70%エタノールを塗布/スプレーし、きれいなはさみと鉗子を使用して下腹腔を露出させます。</li>
	<li>精巣上体脂肪パッド(近位/精巣に付着)を見つける。精巣と上体脂肪パッドの間の接合部を鈍い鉗子で保持し、優しく引っ張って上体脂肪パッドを解放する。</li>
	<li>5 mL の HBSS ではさみと精巣上体脂肪パッドを使用して切断して精巣を取り除き、50 mL の円錐管で 15 分間の温度で BSA を補います。</li>
	<li>遠心分離機 150 x g で 4 °C で 7 分間</li>
	<li>50 mL円錐管から浮遊精液脂肪パッドを取り、きれいなはさみを使用して組織を細かくミンチ。</li>
	<li>200 μL コラゲナーゼ 2% 溶液を 13 mL の培養チューブで 3.8 mL の HBSS に加えます。ひき肉を加え、37°Cで1時間5rpmで回転インキュベーターにインキュベートします。 回転インキュベーターの代わりに、37°Cでの細胞培養インキュベーターを、時折攪拌して使用することができる。</li>
	<li>内容物全体を50 mL円錐形チューブに移し、10mLの中和媒体を加えます。チューブを2~3回反転して軽く混ぜます。</li>
	<li>70 μmのセルストレーナーを取り付けた別の 50 mL 円錐形チューブを準備します。細胞のストレーナーを使用して、消化された組織を新しいチューブにフィルターします。</li>
	<li>フロースルーを15mLの円錐形チューブに移し、遠心分離機を350 x g で4°Cで10分間移動します。</li>
	<li>上清を慎重に取り除き、5 mLのDPBSで細胞ペレットを再懸濁します。遠心分離機 350 x g で 4 °C で 10 分間</li>
	<li>上清を慎重に取り除き、50 μLのフローサイトメトリーバッファーを追加します。穏やかにピペットでよく混ぜ、氷の上に細胞を保ちます。細胞ペレットの体積と残留上清の量が少ないため、フローサイトメトリーバッファー内の細胞の総体積は50μL(約100 μL)を超えます。</li>
</ol>抗体標識と蛍光活性化細胞分類(FACS)
<ol><li>穏やかなピペットで細胞をよく混合した後、細胞懸濁液の54 μLを1.7 mLマイクロ遠心チューブに移します。6 μLのFcRブロッキング試薬を加え、軽くピペットでよく混ぜます。4°Cで10分間インキュベートします。54 μLを取った後に残った細胞を保存し、ステップ3.5およびステップ3.6で未染色コントロールとして使用するために氷の中に保管します。</li>
	<li>ステップ3.1のインキュベーション中に、抗Sca1-APC、抗テル119-FITC、抗CD31-FITC、抗CD45-FITC抗体をマイクロ遠心チューブに組み合わせて抗体カクテルを調製します。穏やかなピペットでよく混ぜ、光から保護された氷の上に保ちます。</li>
	<li>FcRブロッキング試薬を10分間インキュベートした後、40 μLの抗体カクテルを加え、穏やかなピペットでよく混ぜます。4°Cで10分間インキュベートします。</li>
	<li>染色された細胞に、1 μg/mL の DAPI を加えたフローサイトメトリーバッファーを 500 μL 加えます。セルストレーナースナップキャップを用いた5mL試験管を使用して、穏やかなピペットとフィルターセルでよく混ぜます。細胞を氷の上に置いておきなさい。</li>
	<li>ステップ3.1の残りの細胞に500μLのフローサイトメトリーバッファーを加え、穏やかなピペットでよく混ぜます。5 mL の試験管を使用して、セルストレーナー スナップ キャップを使用してセルをフィルター処理します。これらの細胞を氷の上に保管し、次の手順で未染色のコントロールとして使用します。</li>
	<li>FACS分析または選別器具に染色された細胞と未染色の制御を取ります。<ol>
<li>図 1に示すギャティング戦略を使用して、APC+/FITC-/DAPI-母集団を特定し、分離します。これらの細胞は、生存可能なSca1+ /Ter119-/CD31- /CD45- 細胞を表す。残骸と細胞の凝集体は、前方(FSC)および側面散布(SSC)プロットを使用して枯渇します。次に、DAPI-母集団がゲートされ、続いてAPC+/FITC-集団のゲーティングが続きます。不染色コントロールは、ギャティングパラメータの設定を支援するために使用する必要があります。</li>
		<li>細胞を1.5 mLマイクロ遠心分離チューブ内のフローサイトメトリーバッファーの500 μLに回収します。今後、汚染を最小限に抑えるために、すべての手順を無菌条件下で行う必要があります。1つのマウスから約50,000~10万個の細胞が採取されます。</li>
	</ol>
</li>
	<li>細胞の生存率を評価するために、ヘモサイトメーターを使用して細胞を数える。細胞凝集体はさらなる下流分析を妨げる可能性があるため、細胞凝集体の存在も評価され、これらの凝集体の存在を最小限に抑えます(生存可能な細胞数の5%)。</li>
</ol>

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著者らは開示するものは何もない。

単一細胞RNAシーケンシング(scRNA-seq)は、単一細胞レベルで多様な細胞集団を同時に研究する強力なツールとして急速に牽引力を得ています。サンプル調製と高スループットシーケンシングに関連するコストが高いため、実験の成功の可能性を高めるためには、細胞入力(高い生存率と純度)を最適化することが不可欠です。一部の細胞調製プロトコルは、FACSソート14を使用せず...

脂肪組織は、エネルギー代謝に重要な役割を果たしています。.過剰なエネルギーは脂質の形で貯蔵され、脂肪組織は栄養状態およびエネルギー需要に応じて有意な膨張または引き込みが可能である。脂肪組織の拡張は、脂肪細胞サイズの増加(肥大)および/または脂肪細胞数の増加(過形成)から生じる可能性があります。後者のプロセスは、脂肪前駆細胞1,2の増殖と分化によって厳しく調節される。肥満の間、脂肪組織は過度に拡大し、低酸素症、炎症、インスリン抵抗性を含む組織機能不全は、しばしば3、4を発症する。これらの合併症は高血圧、糖尿病、心血管疾患、脳卒中、および癌5を含む多くの慢性疾患への危険因子である。したがって、制御されていない脂肪組織の拡張を制限し、脂肪組織の病理を緩和することは、生物医学研究の最優先事項である。脂肪組織の拡張中に、生体脂肪組織由来幹細胞(ASC)は増殖し、順次に前駆細胞(コミット前駆細胞)に増殖し、次いで成熟脂肪細胞に分化する。最近の単細胞RNAシーケンシング(scRNA-seq)研究は、これらの脂肪前駆細胞(APCおよび予後細胞)集団が実質的な分子および機能的ヘテロジニアリティ7、8、9、10、11、12を示していることを示している。例えば、ASCは、減少した有数分化能力を示す一方で、より高い増殖および拡張能力を示す一方で、前置細胞7と比較して。ASCとプリデポサイト集団内でさらなる分子差が報告されるが、これらの違いの機能的関連性は依然として不明である7。これらのデータは、脂肪の前駆細胞プールの複雑さを強調し、これらの重要な細胞集団をよりよく理解し、操作するためのツールを開発し、標準化する必要性を強調しています。
このプロトコルは、単一細胞研究(scRNA-シーケンシング)および細胞培養を含む、敏感な下流分析に適したマウス上端体脂肪パッドからの高生存率Sca1+脂肪前駆細胞集団の分離を詳述する。上端体脂肪パッドの単離および解離は、前述の7,13と同様に、単離されたAPCの生存率を向上させるわずかな改変を伴って行った。簡単に言えば、上端体脂肪パッドからの解離細胞は、Sca1に対する抗体、ASCおよび予読細胞6、7、および他の系統(Lin)マーカーの両方に対する抗体で染色される:Ter119(エリスロイド細胞)、CD31(内皮細胞)、およびCD45(白血球)。生存可能なSca1+/Ter119-/CD31-/CD45-/DAPI-細胞は、蛍光活性化細胞分類(FACS)によってソートされます。重要なことに、このプロトコルは、ASCおよび予後細胞内の機能的に不均一な亜集団を同定した最近の単一細胞RNAシーケンシング研究で報告された、実行可能なSca1+ /Linの脂肪化前駆細胞の分離および分析に成功して検証された。

<table border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="799">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1.7 mL microcentrifuge tube</td>
			<td style="width:181px;">VWR</td>
			<td style="width:119px;">87003-294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">13 mL culture tube</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-809-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">15 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">188 271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">5 mL test tube with cell strainer snap cap</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">08-771-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">50 mL conical tube</td>
			<td style="width:181px;">Greiner Bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">70 &#181;m cell strainer</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD31-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-CD45-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Sca1-APC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-102-833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Anti-Ter119-FITC antibody</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-112-908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">BSA</td>
			<td style="width:181px;">Gold Biotechnology</td>
			<td style="width:119px;">A-420-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Collagenase type II</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17101-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">DAPI</td>
			<td style="width:181px;">Thermo Fisher</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">F-12 medium</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FcR blocking reagent</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Hanks&#39; balanced salt solution (HBSS)</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Horse serum</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Penicillin-streptomycin</td>
			<td style="width:181px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Propidium iodide solution</td>
			<td style="width:181px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

肥満および代謝障害(糖尿病、心臓病、癌など)は、すべて劇的な脂肪組織リモデリングに関連している。組織に住む脂肪前駆細胞(APC)は脂肪組織恒常性において重要な役割を果たし、組織病理に寄与し得る。単一細胞RNAシーケンシングや単細胞プロテオミクスを含む単細胞解析技術の使用が増え、集団または組織全体の変化の中で個々の細胞発現変化の前例のない解決を可能にすることで、幹細胞/前駆細胞分野を変革しています。本稿では、マウスの上端体脂肪組織を解剖し、単一の脂肪組織由来細胞を分離し、蛍光活性化細胞選別(FACS)を実行して、実行可能なSca1+/CD31 - /CD45-/Ter119- APCを濃縮するための詳細なプロトコルを提供します。これらのプロトコルにより、研究者は単一細胞RNAシーケンシングなどの下流解析に適した高品質のAPCを準備することができます。

この実験では、生後4ヶ月の雄FVBマウスを用いた。FSC/SSCプロットを使用して破片およびダブレットを除外した後、生存可能な細胞(DAPI- 母集団)をゲートし、続いてAPC+/FITC- 母集団の選択を行った(図1)。DAPI、APC、およびFITCのゲートは、未染色の制御に基づいて描かれました。ギャティング戦略を 図 1に示します。
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Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

蛍光活性化細胞選別を用いて、マウス上体脂肪パッドから生存性の高い脂肪前駆細胞を単離する簡単な方法を提示する。

マウス上端体脂肪組織からの脂肪前駆細胞の調製

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

preparation-adipose-progenitor-cells-from-mouse-epididymal-adipose

Research

JoVE Journal

Biology

5.5K ビュー.  Mayo Clinic. 蛍光活性化細胞選別を用いて、マウス上体脂肪パッドから生存性の高い脂肪前駆細胞を単離する簡単な方法を提示する。

ビデオ: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

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

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

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

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

生物学

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

ヒト大動脈血管周囲脂肪前駆細胞の分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

多様なマウス脂肪デポの同定と解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

血管周囲脂肪組織による変調に焦点を当てた孤立腸間膜動脈を用いた血管緊張応答性の評価

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

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

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

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

アカゲザル脂肪由来幹細胞の単離・増殖・分化

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

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 ...

脂肪組織由来幹細胞の単離と特性評価

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

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

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

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

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

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

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.