この研究は生物によって支えられた &#38; 医療技術開発プログラム国立研究財団 (NRF) の &#38; 韓国の政府によって資金を供給 (MSIP &#38; MOHW) (第 2016M3A9B6904244)。私たちもエキソソームの浄化の薬用 Bioconvergence 研究センターのメンバーに感謝します。

1 ブロックの準備、断面、染色や、エキソソームのイメージング
<ol>
	<li>1.5 h23100,000 × g で遠心分離によって HCT116 細胞の培養上清からエクソソームをペレットします。培養上清を削除し、1 ml の 0.1 M ナトリウム cacodylate 溶液 (pH 7.0) 4 ° C で 1 時間で 2.5% グルタルアルデヒドの精製エキソソーム ペレットを慎重に修正0.1 M ナトリウム cacodylate 4.28 g ジメチルアルシン酸 160 mL の蒸留水 (DW) を解散します。0.1 M HCl を蒸留水 200 mL までと 7.4 に pH を調整します。</li>
	<li>定着剤を削除し、室温で 1 ml の 0.1 M ナトリウム cacodylate バッファーのペレットをすすいでください。10 分を持続させる各変更で 3 回を繰り返します。</li>
	<li>4 ° C で 1 時間 2% オスミウム四酸化の 1 ml サンプルを修正後</li>
	<li>固定を外し、すすぎ 3 回 0.1 M ナトリウム cacodylate バッファー 10 分毎。</li>
	<li>傾斜アセトン シリーズで 10 分間インキュベート (50%、60%、70%、80%、90%、95%、100%、それぞれ)、シェーカーで。</li>
	<li>アセトンを取り外して 30 分間 3:1 アセトン: 低粘度埋め込み混合溶液を孵化させなさい。エキソソーム ペレットは、チューブです。</li>
	<li>メディアを取り出して 1:1 アセトン: 低粘度埋め込み混合媒体を追加し、30 分間インキュベートします。</li>
	<li>メディアを取り出して 1:3 アセトン: 低粘度埋め込み混合媒体を追加し、30 分間インキュベートします。</li>
	<li>メディアを取り出して、100% 低粘度混合物を埋め込み、室温で一晩インキュベートを追加します。</li>
	<li>純粋な低粘度 65 ° C で 24 h の埋め込み型と焼くを使用して混合物を埋め込むサンプルを埋め込む</li>
	<li>超ミクロトームで 60 nm の厚さでセクションを準備します。</li>
	<li>20 分間の 2% 酢酸ウラニウム二重染色と 10 分のクエン酸をリードします。 レイノルズ鉛ソリューション 1.33 g 鉛硝酸とクエン酸ナトリウムの 1.76 g を 50 ml の蒸留水の合計に追加します。</li>
	<li>80 で透過型電子顕微鏡下でグリッドを観察 kV。</li>
	<li>「取得」をクリックし、、クリックして「ファイル」と"Save as"80 で電子顕微鏡 CCD カメラ システムの kV。露光時間の自動設定に従います。</li>
</ol>
2. セクションおよびイメージング (図 1) の免疫染色
<ol>
	<li>超ミクロトームを使用して 60 nm の超薄切片を切り、ニッケル グリッドを収集します。</li>
	<li>50 μ L の滴無料アルデヒド グループをクエンチする 10 分の 0.02 M のグリシンのグリッドを孵化させなさい。</li>
	<li>リンス DW の 100 μ L で 3 回それぞれ 1 %bsa を含む PBS で 1 時間室温で 10 分間加温。</li>
	<li>50-100 μ L 滴 KRS 抗体24 (0.1 %bsa を含む PBS で 1: 100) アンチのグリッド間インキュベート 1 時間に応じて、このステップべきである 4 ° C で一晩。</li>
	<li>10 分の 0.1 %bsa を含む PBS の 5 別滴 (50 μ L) でグリッドを洗います。</li>
	<li>2nd抗体 (抗うさぎ IgG が 0.1 %bsa を含む PBS で 10 nm の金粒子 (1: 100) に共役) 1 h の滴にグリッドを転送します。</li>
	<li>5 洗浄グリッドは各 10 分 0.1 %bsa を含む PBS の滴 (50 μ L) を区切ります。</li>
	<li>ダブル-染色暗い条件の下で 20 分間酢酸 2% ウランとレイノルドの鉛クエン酸 10 分間それぞれ。</li>
	<li>「取得」をクリックし、、"ファイル"と"Save as"CCD カメラ システム 80 で電子顕微鏡の下でをクリック kV。露出時間は続いた自動設定です。</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56482/56482fig1.jpg"> 
図 1: サンプル調製と染色のプロセス。(A)二重固定エキソソームが脱水および混合樹脂を埋め込み低粘度で浸透します。(B)の樹脂を埋め込みます。(C)ダイヤモンド ナイフで超薄いセクション。(D)免疫金ラベルを設定します。超薄切片のグリッドは、湿らせたペーパー タオルに配置されますのパラフィルムで液滴に置かれます。各セクションは 50-100 μ L 抗体の液滴と孵化し、バッファーで洗浄します。(E)酢酸ウラニウム染色を二重になりクエン酸。蓋は、重金属染色用アルミ箔で覆われています。(F)は染色液はろ紙によって削除されます。<a href="//ecsource.jove.com/files/ftp_upload/56482/56482fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
3. 負の染色
<ol>
	<li>グロー放電グロー放電によって 1 分ホルムバール/炭素薄膜塗膜 200 メッシュ銅 EM グリッド。</li>
	<li>2 %1 ml 精製エクソソームを修正パラホルムアルデヒド (PFA) 5 分。 注意: パラホルムアルデヒド煙、有毒です。すべての作業は、換気の発煙のフードで行われるべき。</li>
	<li>グリッド上の 5-7 μ L エキソソーム懸濁液ソリューションを読み込むし、1 分間インキュベートします。エキソソームの濃度が高すぎる場合は、1/2 - 1/5 の濃度を薄めます。</li>
	<li>すぐに注射器による EM グリッド面フィルター 1% ウラニル酢酸 (UA) ソリューション 〜 20 滴で染色します。</li>
	<li>ろ紙をグリッド エッジに連絡してグリッドに過剰な UA ソリューションを削除します。</li>
	<li>すぐに水を一滴を持つグリッドをすすいでください。このステップは、余分な染色液が削除されます。</li>
	<li>ピンセットでグリッドをテーブルに置くし、室温下で 10 分間乾燥する培養皿で部分的にグリッドをカバーします。</li>
	<li>80 で TEM による未来の観察のための EM グリッド ボックスのグリッドを保存 kV。</li>
</ol>
4 是非、全体のマウント
<ol>
	<li>グロー放電ホルムバール/炭素薄膜フィルム コーティング 200 メッシュ銅 EM グリッド 30 s。</li>
	<li>2 %1 ml 精製エクソソームを修正パラホルムアルデヒド (PFA) 5 分。 注意: パラホルムアルデヒド煙、有毒です。すべての作業は、換気の発煙のフードで行われるべき。</li>
	<li>エキソソーム ソリューションを固定グリッド上 5-7 μ L を読み込むし、インキュベート pbs 100 μ L に 5 分すすぎ 3 回各 10 分。</li>
	<li>無料アルデヒド グループをクエンチする 10 分の 0.05 M のグリシンの 50 μ L でグリッドを扱います。</li>
	<li>30 分のブロック バッファー (1 %bsa を含む PBS) のドロップにグリッドを転送します。</li>
	<li>50-100 μ L 抗 PD L1 抗体 (0.1 %bsa を含む PBS で 1: 100) でグリッドを 1 時間に応じて、このステップべきである 4 ° C で一晩インキュベートします。</li>
	<li>10 分の 0.1 %bsa を含む PBS の 5 別滴 (50 μ L) でグリッドを洗います。</li>
	<li>グリッドを 1 h の 2nd抗体のドロップに転送します。</li></ol>0.1 %bsa を含む PBS で 1: 100 希釈 9-11 nm の金粒子標識抗マウス IgG。
	<li>10 分の 0.1 %bsa を含む PBS の 5 別滴 (50 μ L) でグリッドを洗います。DW の 2 独立した滴 (50 μ L) とグリッドを洗います。2% 酢酸ウラニウム 3.8 3.4 から前述の否定的な汚損を実行します。</li>
	<li>80 で TEM による未来の観察のための EM グリッド ボックスのグリッドを保存 kV。</li>

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この記事では、詳細なエキソソームの構造を観察し、その特定の蛋白質の分類のためのプロトコルを示します。否定的な汚損はエキソソーム17をイメージングに最適な方法として考えられています。この手法は、エクソソームのカップ状の構造を示しています。しかし、このカップの形状は乾燥プロセスによって発生するアーチファクトのフォームです。低温電子顕微鏡 TEM ...

細胞小胞 (Ev) は、細胞から分泌される脂質二分子膜ベシクル、30、300 nm 間のサイズの範囲します。EVs は、ホジキン病1患者の小胞からの証拠と、1978 年に最初に報告されました。これらの小胞はサイズ 40 に 120 ナノメートルと報告されました。1980 年に、EVs が凝固系と血栓症、癌患者2の血管内血栓の形成に関与していることが報告されました。30 年後、EVs は、浸潤、免疫回避、および血管新生3を促進するために重要な要因であること報告されています。さらに、EVs 機能は、炎症、免疫障害、神経疾患、がん4の領域における細胞間相互作用のレギュレータとして研究されています。EVs は、マイクロ Rna5mRNA、タンパク質など特定の生体分子を含む、診断および治療の潜在的な応用解析6,7されています。EVs は、エクソソーム、prostasomes、oncosomes、dexosomes、微粒子、promininosomes、argosomes、およびエキソソームのような小胞細胞の起源および生物学的機能3によってを含むサブグループから成るカテゴリです。さらに、彼らの器官を基に、これらの EVs に分かれます (小屋機構の解明、100-1000 nm) 機構の解明およびエクソソーム (30-300 nm)8,9。これらのうち、免疫応答10、癌11,12、および伝染病13セル コミュニケーターとして、エキソソームを報告されています。
早期診断マーカーとしてエクソソームの関心が急速に高まっていると精製とエクソソームの性質は、分子イメージング技術と同伴が必要があります。その起源と機能を14、に応じて、エクソソームの形態とさまざまなサイズは、電子顕微鏡などの高解像度の顕微鏡検査の技術によって区別できます。ほとんどエクソソームいた否定的なステンド グラス透過電子顕微鏡 (TEM)15,16,17、可視化し、これらの結果は、全台紙の小胞の特定の蛋白質の反応によって確認されました。18. いくつかの研究グループは、走査電子顕微鏡、原子間力顕微鏡19、20、および低温電子顕微鏡 TEM21,22によって構造を報告しています。ただし、これらのテクニックはエキソソーム構造を調べる場合に役立ちますが、彼ら、エキソソームの内側にある特定の蛋白質の位置観測のために十分ではありません。したがって、特定の蛋白質のラベリングとエクソソームをイメージングするためのプロトコルを導入しました。我々 は、ブロック準備、極薄切片および免疫染色、エキソソームのタンパク質の詳細な場所を判別するに適用されます。否定的な汚損と全体マウント免疫染色は、昔から、エキソソームの特性と比較しました。

<table><tbody><tr><td>Glutaraldehyde</td><td>EMS</td><td>16200</td><td></td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>EMS</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide 1 g crystal</td><td>EMS</td><td>19100</td><td>Use only in fume hood</td></tr><tr><td>acetone</td><td>JUNSEI</td><td>1B2031</td><td></td></tr><tr><td>Spurr medium</td><td>TED PELLA</td><td>18108</td><td></td></tr><tr><td>Ultra-microtome</td><td>Leica</td><td>UCT</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Lead citrate</td><td>EMS</td><td>17900</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>H7600</td><td></td></tr><tr><td>nickel grid</td><td>EMS</td><td>G200-Ni</td><td></td></tr><tr><td>Copper grid</td><td>EMS</td><td>G200-Cu</td><td></td></tr><tr><td>glycine</td><td>SIGMA</td><td>022K5404</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>SIGMA</td><td>P4417</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Aurion</td><td>25557</td><td></td></tr><tr><td>1st Antibody</td><td></td><td></td><td>purification in manually&amp;nbsp;</td></tr><tr><td>Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td><td>BioLegend</td><td>329710</td><td>Whole mount Immumogold</td></tr><tr><td>Purified Mouse IgG2b, &amp;kappa; Isotype Ctrl</td><td>BioLegend</td><td>401202</td><td>Whole mount Immumogold (Control)</td></tr><tr><td>2nd Anti-mouse igG conjugated 9-11 nm gold particle</td><td>sigma</td><td>G7652</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr><tr><td>Glow discharger</td><td>JEOL</td><td>JFC1100E</td><td></td></tr><tr><td>Carbon grid</td><td>EMS</td><td>121119</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19210</td><td></td></tr><tr><td>Formvar carbon coated Copper Grid (200 meh)</td><td>EMS</td><td>FCF200-CU</td><td></td></tr><tr><td>Formvar carbon coated&amp;nbsp; Nickel Grid (200 mesh)</td><td>EMS</td><td>FCF200-NI</td><td></td></tr></tbody></table>

sample preparation and imaging of exosomes by transmission electron microscopy

エクソソームは体液によって分泌されるナノ細胞小胞であり、それらを分泌する細胞の特性を表すために知られています。分泌小胞の形態と内容は、セルの動作または生理学的状態、たとえば細胞増殖、移行、胸の谷間と死を反映します。エクソソームの役割のサイズに非常に依存し、エクソソームのサイズ、30 ~ 300 nm。エキソソーム イメージングのため最も広く使用されている方法が否定的な汚損、その他の結果は、クライオ電子顕微鏡、走査電子顕微鏡、原子間力顕微鏡に基づいていますが。典型的なエキソソームの形態の否定的な汚損によって評価はカップの形状が、さらに詳細はまだ分からない。エキソソームの構造研究を特徴は医学・薬学分野の特定の必要です。したがって、関数に依存した形態はエキソソームの詳細構造の特定の蛋白質のラベル付けなどの電子顕微鏡検査の技術によって検証すべき。詳細な構造を観察するには、エクソソームの負の染色像と極薄, 切片画像を比較しました。このプロトコルでは、私達は否定的な汚損、全体マウント免疫染色、ブロック作製、薄セクション、および免疫金ラベルを含むエクソソームのイメージングのための透過型電子顕微鏡を提案します。

現在、エクソソームは透過電子顕微鏡によるサイズと形状のカテゴリに分類されます。図 2に示します負エキソソームと全体のマウント ・ ステータスに免疫標識エキソソームを染色します。図 3は、薄切片法後断面エキソソームと免疫標識エクソソームを示します。免疫金は特定の蛋白質の抗体を用いて染色は、積極的に?...

Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

このプロトコルでは、否定的な汚損、詳細な構造と免疫金エクソソームの特定の蛋白質の位置を判断するための極薄断面透過型電子顕微鏡に必要な様々 なテクニックについて説明します。

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

sample-preparation-imaging-exosomes-transmission-electron

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

38.7K ビュー.  Asan Medical Center. このプロトコルでは、否定的な汚損、詳細な構造と免疫金エクソソームの特定の蛋白質の位置を判断するための極薄断面透過型電子顕微鏡に必要な様々 なテクニックについて説明します。

ビデオ: Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子間力顕微鏡による細胞外小胞のイメージング

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

生化学

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

ランダムコニカルチルト方式を使用したエキソソーム複合体の単粒子の電子顕微鏡再構成

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

生物学

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Nanoplasmon の散乱と低倍率の顕微鏡イメージングによる腫瘍由来エキソソームの定量化

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

生物工学

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

医学

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含有エキソソームの単離と解析

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

バイオメディカルアプリケーションにおけるエクソソームの持続的送達のためのマイクロニードルパッチ

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.

著者スポットライト:エクソソームの送達と安定性を強化するための革新的なマイクロニードルベースの戦略

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.

エクソソームのローディングとデリバリーのためのマイクロニードルパッチの作製と特性評価

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3次元バイオリアクター培養による滑液間葉系幹細胞由来エクソソームの大規模調製

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

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原子間力顕微鏡による細胞外小胞のイメージング

ランダムコニカルチルト方式を使用したエキソソーム複合体の単粒子の電子顕微鏡再構成

Nanoplasmon の散乱と低倍率の顕微鏡イメージングによる腫瘍由来エキソソームの定量化

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

RNA含有エキソソームの単離と解析

エキソソームの定量化とサイズの測定のための革新的方法

エクソソームのローディングとデリバリーのためのマイクロニードルパッチの作製と特性評価

エクソソームのローディングとデリバリーのためのマイクロニードルパッチの作製と特性評価

エクソソームのローディングとデリバリーのためのマイクロニードルパッチの作製と特性評価

3次元バイオリアクター培養による滑液間葉系幹細胞由来エクソソームの大規模調製