Name: isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue
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著者はベッキー マルケス、センタラ肥満センター、患者スクリーニングと同意のプロセスに彼女の援助のための医療コーディネーターを認識したいです。この研究は、Anca + Dobrian に R15HL114062 によって支えられました。

東バージニア医学校制度のレビュー ボード委員会承認研究および研究で使用されるひと脂肪組織サンプルのコレクション。通知の書面による同意は、患者から採取しました。
1. バッファー、メディア、および計測器の準備
<ol><li>Krebs Ringer Bicarbonate-Buffered ソリューション (KRBBS) を準備: 塩化ナトリウム 135 ミリメートル、5 mM 塩化カリウム、硫酸マグネシウム 1 mM、0.4 mM ?...

<ol>
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本稿の焦点は、分離、拡張および CD31 cd34 陽性 + + ひと脂肪組織内臓や皮下のデポから血管内皮細胞の脂肪分化誘導方法論を提供することです。
齧歯動物または CD31 抗体、蛍光ラベルまたは磁気ビーズ18,に結合を使用して主にテクニックを使って人間の様々 な血管ベッドから血管内皮細胞の隔離のための方法論が報告されています。<sup class="xref...

最近の証拠は、マウスとヒトでは、白またはベージュ/ブラウン脂肪1,2,3に脂肪組織の血管に存在するセルのサブセットを区別できますを示しています。このような細胞の表現型の中間表現型4,5,6、7のスペクトルや平滑筋/周皮細胞、血管内皮細胞を支持する証拠との論争の主題であります。この方法論の開発の範囲は、CD31 cd34 陽性 + + 肥満人間から別の脂肪質のターミナルから分離した内皮細胞の脂肪分化能をテストするためだった。文献の他の研究は、潜在的な合計の間質血管の分数または知られている脂肪細胞の前駆細胞の2,8,9の脂肪細胞に着目します。現在既存の技術は、薬配信10特に脂肪組織の血管内皮細胞をターゲットすることができますので、このような細胞の脂肪分化を受ける可能性を理解白またはベージュの脂肪細胞へ誘導重要です。将来の目標とされた療法。
別のグループは、ひと脂肪組織11,12,13から血管内皮細胞を分離するサロゲートとして CD31 と CD34 マーカーの組み合わせを報告しました。通常、2 つの連続した手順と磁気ビーズを用いた正の選択を使用して分離が実行されます。本報告では、CD31 プラスチック ビーズと組み合わせて CD34 陽性磁気ビーズを用いた immunoseparation に利用されました。典型的な石畳の内皮形態の保全に関して逐次磁気 immunoseparation に優れたこの手法がわかった。また、脂肪の 1-2 g ほどから始まって拡大と脂肪細胞の誘導に必要な十分な細胞を生成することができました。皮下脂肪の小さなサンプル生検は、下流用電池の必要な量を生成するのに十分です。この面は可能性のある重要な特に場合は、このメソッドは人間の脂肪分化誘導に対する反応性のスクリーニングに使用されます。
文献で報告された他のシステムとは異なり、この方法は CD31 cd34 陽性 + + 細胞の脂肪分化誘導のための 2 つの成分を利用: ppar γ アゴニスト-ロシグリタゾン — とヒトインスリンです。重要なは、インスリンの使用量は、人間14post-absorptive インスリンを循環通常/高の範囲内にあります。カクテルの誘導に対応する能力と、細胞の in vitro、Akt のリン酸化による測定のインスリンに対する反応性の程度は相関しなかった。興味深いことに、この誘導のカクテルや実験条件を使用して、白とベージュ/ブラウン細胞のミックスは、サイズと分子マーカーの発現と細胞内脂質液滴の数によって決定された得られました。(白とベージュ)、細胞の表現型の定量的評価と一緒にこの簡単かつコスト効果の高い誘導プロトコルは、差別化されたベージュのバランスを変更することができます潜在的エージェントのスクリーニング: 白色脂肪細胞です。
このメソッドは、ひと脂肪組織の血管内皮前駆細胞の脂肪細胞形成の基になるメカニズムを理解するためトランスレーショナル アプローチを提供します。この特定の分離/差別化手法を使用して、捜査官は様々 な経路の細く、肥満の人間の様々 な脂肪質のターミナルから血管内皮細胞のサブセットの脂肪細胞の形成を担当に問い合わせることができます。

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

肥満は脂肪細胞の肥大によって主に脂肪組織の大規模な改造を伴います。極端な脂肪細胞成長は、インスリン、ローカル低酸素症および炎症に悪い応答が返される前駆細胞から機能的な白い脂肪細胞の分化を刺激し、脂肪細胞の人口の根本的な肥大を防ぐことができるしの削減とともにその結果、脂肪代謝の健康を改善できます。炎症。また、ベージュ/ブラウンの脂肪細胞の分化を刺激しと、全身のエネルギー消費量を増やすことができる減量、その結果します。このアプローチは、肥満 2 型糖尿病と心血管疾患などの併存の開発を防ぐことができます。
本稿では、分離、拡張、および共同 CD31 と CD34 のマーカーを表現するひと脂肪組織の血管内皮細胞のサブセットから白とベージュの脂肪細胞の分化をについて説明します。メソッドは、比較的安価ですし、労働集約的ではないです。人間の脂肪組織へのアクセスを必要として皮下のデポはサンプリングに適しています。このプロトコルの新鮮な脂肪組織は、病的肥満者からサンプル [ボディマス指数 (BMI) > 35] 肥満手術の手順中に収集されます。間質血管の一部から順次 immunoseparation を使用して、十分な細胞は、脂肪の 2-3 g ほどから生成されます。これらの細胞文化 10-14 日間で拡大することがでくことができますが, 凍結保存、通路 5-6 まで継と、脂肪細胞のプロパティを保持します。細胞は、脂肪細胞の ppar γ アゴニスト ロシグリタゾンとヒトインスリンの組み合わせを使ってカクテルを 14 日間扱われます。
この方法は脂肪の血管内皮細胞、脂肪細胞応答を駆動する分子メカニズムに関する実験についての証明書を取得するため使用ことができますまたは、脂肪細胞を強化することができます新しい薬物をスクリーニングするため応答指示ホワイトのどちらかベージュ/ブラウン脂肪細胞分化。小さな皮下のバイオプシーを使用して、この手法を使用して肥満と併存の治療のためベージュ/茶色と白色脂肪細胞を刺激することを目的とする臨床試験のための非レスポンダー科目を画面できます。

私達のプロトコルは CD31 cd34 陽性 + + ひと脂肪組織別拠点から血管細胞の脂肪分化能を決定するためのアプローチを提供することを目的します。簡単なフローチャート図は、図 1 aに表示されます。CD34 の発現細胞の正の選択を使用して最初のステップ > 95 %cd34 陽性細胞新鮮単離細胞 (図 1 a) の人口で起因します。重要?...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

白とベージュの脂肪細胞脂肪組織血管前駆細胞からの分化は、肥満の代謝改善の可能性を負いません。CD31 cd34 陽性 + + 人間の脂肪から内皮細胞分離および後続の in vitro拡張と白とベージュの脂肪細胞への分化のプロトコルについて述べる。いくつかのダウン ストリーム アプリケーションを説明します。

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

This method can help answer key questions in the obesity field, such as identifying the key factors and mechanisms that prevent a robust adipogenic response during adipose tissue remodeling. The main advantage of this technique is that it allows isolation of CD34+CD31+endothelial cells from human adipose tissue starting with small amounts of tissue. We first had the idea for this method when we wanted to identify cells in human adipose vasculature that respond to adipogenic stimulation.Visual demonstration of this method is critical, as the cell isolation steps are difficult to practice due to the oftentimes limited access to fresh human adipose tissue. Collect human omental and subcutaneous adipose tissue from human subjects undergoing bariatric surgery. Immediately after tissue extraction, keep the human omental and subcutaneous adipose tissue in separate vials containing Hank's buffered salt solution with 50 micrograms per milliliter of penicillin streptomycin at room temperature.Once the sample arrives at the lab after the tissue extraction, carefully clean and remove fibrotic and cauterized sections of the tissue by using 70%ethanol-swabbed scissors and tweezers. Weigh the adipose tissue and partition it into five gram aliquots for the collagenase digestion. Use two pairs of scissors to finely mince five grams of the adipose tissue in five milliliters of a collagenase solution in a scintillation vial at room temperature.Add an additional 10 milliliters of collagenase solution to the minced tissue for 15 milliliters total. Digest the samples for one hour at 37 degrees Celsius in a water bath with continuous shaking. Following digestion, pour the samples into the 20 milliliter blunt end syringe.Filter the sample through 250 micron nylon mesh into the 50 milliliter conical tube to separate adipocytes and stromal vascular cells from undigested tissue. Wash out the scintillation vial with 10 milliliters of KRBBS, and pour the wash through the filter to collect residual cells before incubating the filtered samples for five minutes at room temperature. Then, utilize a 20 milliliter syringe and a 20 gauge by six inch pipetting needle to remove stromal vascular fraction layer and transfer it to a clean 50 milliliter conical tube.Add 10 milliliters of KRBBS and allow the samples to sit on the bench for five minutes at room temperature. Keep the stromal vascular fractions from both depots on ice for further processing. Spin the stromal vascular fractions at 500 times G for five minutes at 4 degrees Celsius.Remove the samples from the centrifuge and carefully pour off the supernatant, keeping the pellet containing the stromal vascular fraction cells. Gently re-suspend the cell pellets in five milliliters of PBS. After spinning the samples again, remove the supernatant, re-suspend the cells in one milliliter of PBS, and count the cells.Pellet the samples for a third time, and re-suspend the pellet in 100 microliters of CD34 isolation buffer. Add 10 microliters of CD34 cocktail and incubate the sample for 15 minutes at room temperature. Then add five microliters of magnetic beads and incubate the sample for 10 minutes at room temperature.Next, add 2.5 milliliters of CD34 isolation buffer to each sample, and place the samples into the magnet for five minutes at room temperature. Invert the magnet for five seconds to pour off the isolation buffer. After repeating this process four times, remove the tube from the magnet and add two milliliters of CD34 isolation buffer.Following another centrifugation, re-suspend the CD34+cell pellets in one milliliter of CD34 isolation buffer and count the cells. Pellet the cells again, and re-suspend the pellet in 250 microliters of Buffer B and 250 microliters of wash buffer. Next, thoroughly re-suspend the CD31 beads by vortexing the tube, then add 20 microliters of CD31 beads to the re-suspended cell pellet and incubate the sample with rocking for 30 minutes at room temperature.Prior to the cell separation, add one milliliter of wash buffer to equilabrate the strainer. Then, apply the prepared mixture of re-suspended cells in CD31 beads to the strainer. Wash the strainer with five milliliters of wash buffer in a circular motion for a total volume of 20 milliliters.Attach the connector to a sterile 50 milliliter conical tube and close the leur lock. After attaching the strainer to the connector, add one milliliter of wash buffer along the wall of the strainer, then add one milliliter of activated Buffer D along the wall of the strainer. Swirl the sample gently, and incubate it for 10 minutes at room temperature.Next, add one milliliter of wash buffer and separate the cells from the beads by pipetting up and down 10 times. Open the leur lock and allow the detached cells to flow into the 50 milliliter conical tube. Wash the strainer 10 times with one milliliter of wash buffer each time.Discard the connector and strainer, and centrifuge the samples at 300 times G for 10 minutes at 4 degrees Celsius. Finally, re-suspend the cell pellet in two milliliters of complete EGM2 media for culture. Grow the cells in six-well tissue culture treated plates with complete EGM2 media in a 37 degrees Celsius, 5%carbon dioxide incubator.Replace the media every three to four days until the cells reach 80 to 90%confluence. Then, seed approximately 100 to 200 thousand cells into six-well plates, ensuring duplicate wells for each depot, and culture them in complete EGM2 media for two days. After two days, aspirate off any growth media.Replace the media with two milliliters of DMEM/F-12 media with 5%FBS and 50 micrograms per milliliter of penicillin streptomycin for the control cells. For the treatment cells, replace the media with two milliliters of adipogenesis media. Incubate the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 13 days replacing the respective media every three to four days.Representative results showing the typical endothelial cobblestone-like morphology of the CD34+CD31+cells after two passages in culture is shown. Also demonstrated is the ability of cells to form tubules when cultured in Matrigel as expected. Uniform uptake of fluorescently labeled acetylated LDL shows the presence of a homogenous cell population with endothelial identity.Shown here is Oil Red O staining of neutral lipid droplets in CD34+CD31+cells following 13 days of adipogenic induction. Representative responses of cells from omental and subcutaneous depots of three different human subjects are shown to illustrate the large variability in response to adipogenic induction between fat depots of the same donor and between donors. This image displays fluorescent intracellular lipid staining in CD34+C31+cells after adipogenic induction for 13 days.The image shows cells with unilocular lipids, multilocular lipids, and no lipid accumulation. Real time PCR shows the expression of the pan adipocyte marker adiponectin and the brown beige markers CIDEA and UCP-1. These results confirm that cells following adipogenic induction express molecular markers consistent with a mixed white beige adipocyte phenotype.Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it is important to remember to use fresh human adipose tissue that was collected and not stored longer than two hours. Following this procedure, other methods like flow cytometry, immunohistochemistry, and RT-PCR can be performed to identify specific subsets of responding cells, and expression of mature adipocyte markers that can be utilized to identify a beige brown adipocyte phenotype.After it's development, this technique paved the way for researchers in the filed of obesity to explore roles of adipose tissue, vascular cells, and adipogenesis in humans. After watching this video, you should have a good understanding of how to isolate, culture, and expand CD34+31+cells. Don't forget that working with human tissues can be extremely hazardous, and precautions, such as bio-safety regulations, should always be taken while performing this procedure.The implications of this technique extend toward therapy of obesity associated comorbidities, such as insulin resistance, because it identifies adipogenic response of cells to a potential therapeutic intervention.

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

免疫・感染学

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation of Adipose Tissue Immune Cells

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

ヒト母体·胎児のインターフェイスから白血球の単離

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

アデノウイルスに感染したヒト正常細胞からのウイルス複製コンパートメントに富むサブ核画分の単離

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

末梢血およびリンパ組織から主な人間の T 細胞のシナプスのインタ フェースの評価

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

ヒトおよびマウスの心筋標本からのマクロファージサブセットおよび間質細胞の分離

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...