Name: Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells
Uploaded: 2024-01-26T14:10:00
Description: Watch this Scientific Journal Video about Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells at JoVE.com

isolation and preparation of endosomes containing magnetic nanoparticles from cultured cells

エンドソーム由来ナノベシクルの磁気的高収率生産の最適化

Extracellular vesicles (EVs) are small vesicles secreted by almost all cells with a size range of 30-150 nm, containing abundant bioactive substances. Depending on the cell of origin, EVs show high heterogeneity, possessing multiple components specific to parent cells1. EVs are released into body fluids and transported to distant sites where they are taken up by target cells for action2, which can be utilized to deliver a wide range of bioactive molecules and drugs for tissue repairing, tumor diagnosis and treatment, and immune modulation3,4. However, other biological nanoparticles (e.g., lipoproteins) and nanovesicles (e.g., EVs derived from non-endosomal pathways) with similar biophysical properties in body fluids inevitably affect EV isolation and purification. To date, ultracentrifugation remains the gold standard for EV isolation, and other isolation methods, including sucrose density gradient centrifugation, ultrafiltration, polyethylene glycol precipitation, chromatography, and immunomagnetic bead isolation, have been developed5. The current bottleneck limiting clinical translation and commercialization of EV therapeutics is the severe lack of isolation techniques that allow for highly scalable and reproducible isolation of EVs6,7,8. Traditional EV isolation techniques (e.g., ultracentrifugation and size exclusion chromatography) suffer from low yield (1 x 107-1 x 108/1 x 106 cells), long production cycle (24-48 h), poor reproducibility of product quality, and require expensive and energy-intensive production equipment that cannot meet the current clinical demand for EVs6.
Exosome mimics (EMs), synthetic surrogates of native EVs, have attracted important attention due to their highly similar structure, function, and scalability in production. The main source of EMs is from the direct extrusion of whole parental cells with continuous sectioning9,10, demonstrating potent biological functions as native EVs11,12. For instance, EMs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) exert similar wound-healing effects as native EVs and are richer in protein composition13. Though EMs derived from whole cells have the biological complexity of EVs, their main drawback is the heterogeneity of products because they are inevitably contaminated by various cellular organelles and cell debris. Protein localization analysis further revealed that EMs derived from whole-cell extrusion contain many non-EVs-specific proteins from mitochondria and the endoplasmic reticulum13. Moreover, most methods for manufacturing EMs still require ultracentrifugation, a highly time and energy-consuming process14. Considering the fact that exosomes are exclusively derived from cellular endosomes, we hypothesized that bioengineered endosome-derived nanovesicles may better recapitulate the biological homology between exosomes and EMs in comparison with the well-established cell membrane-derived EMs produced by whole cell extrusion method14. Nevertheless, the manufacture of endosome-derived nanovesicles is difficult due to the lack of viable approaches.
Clinical studies have been carried out by utilizing EVs as a surrogate of cell-free therapy and a nanoscale drug delivery system for the treatment of various diseases. For instance, EVs derived from bone marrow mesenchymal stem cells have been used to treat severe pneumonia caused by COVID-19 and have achieved promising results. Recently, genetically engineered EVs carrying CD24 proteins have also demonstrated potent therapeutic benefits for treating COVID-19 patients15,16. However, the clinical requirement of EV therapy still cannot be met with traditional isolation methods because of the low yield and cost. This study reports the large-scale production of endosome-derived nanovesicles via a magnetic separation-assisted high-speed homogenization approach. It takes advantage of the endocytosis pathway of MNPs to isolate MNP-loaded endosomes via magnetic separation, followed by high-speed homogenization to formulate endosomes into monodisperse nanovesicles. Since the types of endosomes collected by this protocol are diverse, further in-depth research is still required to establish good manufacturing practices (GMP) in the industry. This novel EM preparation approach is more time efficient (5 min of high-speed homogenization) to obtain nanovesicles homologous to native EVs. It produces exponentially more vesicles from the same amounts of cells than ultracentrifugation, which can be generally applied to various cell types.

著者スポットライト:磁性ナノ粒子を用いたエクソソーム模倣体の大規模再現性のある作製法の開発 

エンドソーム由来小胞の大量生産のための磁気分離支援高速均質化法

In this study, we try to address a critical unmet need in exosome research, which is developing a large-scale and reproducible production method for exosome mimetics. Due to the notion of native evades, exosome mimetics, EMS, has been produced as surrogates by methods such as real exclusion and ultracentrifugation based filtration. Certainly most exosome mimetics are from the direct exclusion of whole cells, which demonstrates minimal biologic functions, but are inevitable contaminated by cell debris and organelles.Extracellular vesicles have attracted significant attention in biomedical research, especially in disease treatment. However, traditional EV isolation techniques like ultrasensitive filtration, inside exclusion, catamental glyphic start from low-yield poor productibility of plow down quality and require expensive and engineer intensive production equipment that cannot meet the clinical demand for EVs. Our study provided a simple and no cost method for scan up production of exosomes mimetics.We take advantage of unique biological finding that magnetic nanoparticles can be only endonized within cell endosomes, not other organelles. Thus, we can formulate the endosomes into uniform nanosescose. This method effectively improve EME and reduces cost.

Watch this Scientific Journal Video about Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells at JoVE.com

Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells  

培養細胞からの磁性ナノ粒子を含むエンドソームの単離と調製

まず、10%FBSおよび5%ペニシリン-ストレプトマイシンを含むDMEM完全培地で6ウェルプレートで細胞を培養します。培養液を摂氏37度、二酸化炭素5%で一晩インキュベートします。翌日、細胞を10ナノモルのポリリジン変性酸化鉄ナノ粒子またはIONPで共培養し、摂氏37度、二酸化炭素5%で12時間インキュベートします。インキュベーション後、ウェルあたり0.5ミリリットルのトリプシンを3分間添加して細胞を消化します。次に、ウェルごとに1ミリリットルの完全培地を追加して、消化を停止します。細胞懸濁液を 1, 000 g で 5 分間遠心分離し、上清を捨ててからペレットを PBS に再懸濁します。次に、ペレットを8ミリリットルの新しく調製した高張溶液に15分間再懸濁します。細胞懸濁液をガラス試験管に移し、ガラスホモジナイザーを使用して、1, 000 RPMで20回のショックを与えて細胞小器官を放出します。ホモジナイズ後、1ミリリットルの細胞懸濁液を1.5ミリリットルの微量遠心チューブに移します。チューブを磁気分離器に1時間入れて、IONP負荷エンドソームを他の細胞小器官や細胞破片から完全に分離します。エンドソームを回収するには、チューブから液体を捨て、3ミリリットルのPBSを加えてエンドソームを再懸濁します。次に、ピペットガンを使用してブローし、磁気フレームの隣のチューブの接触面にある茶色のペレットを再懸濁させます。高速ホモジナイズのために、エンドソーム溶液を15ミリリットルの遠心チューブに移します。アイスボックスをチューブの下に置き、チューブを高速ホモジナイザーに置きます。10ミリリットルのプローブを底に触れずにチューブに入れます。ホモジナイザーの画面で、速度を 140 g に調整し、時間を 5 分に設定します。OKボタンを押してから、スタートボタンを押します。高速ホモジナイズ後、ナノベシクル懸濁液を1.5ミリリットルのチューブに移し、チューブを磁気セパレーターに1時間入れます。最後に、必要なエキソソーム模倣体またはEMを含む液体を、磁気フレームの隣の表面を乱すことなく回収します。NTAおよびTEMで解析したBMSC-EMの形態は、脂質二重層で区切られた典型的なお椀状の小胞状構造を示しました。BMSC-EMと293T-EMは、どちらもネイティブEVと同様の流体力学的直径を持っていました。ウェスタンブロッティングの結果、BMSC-EMはEVと同じタンパク質バイオマーカーを含み、細胞膜の汚染がほとんどないことが示されました。EMSの収率は、超遠心分離法で調製されたネイティブEVよりも有意に高かった。EMのタンパク質組成は、ネイティブEVと似ていました。

isolation-preparation-endosomes-containing-magnetic-nanoparticles

Research

JoVE Journal

Biology

Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, their clinical translation has been limited by the lack of scale-up manufacturing approaches. Therefore, this protocol provides a magnetic separation-assisted high-speed homogenization method for the large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) derived from the endosomes, which have about 100-time higher yield than conventional ultracentrifugation method. In this method, magnetic nanoparticles (MNPs) were internalized by parental cells via endocytosis and were subsequently accumulated within their endosomes. Then, MNPs-loaded endosomes were collected and purified by hypotonic treatment and magnetic separation. A high-speed homogenizer was utilized to break MNP-loaded endosomes into monodisperse nanovesicles. The resulting endosome-derived vesicles feature the same biological origin and structure, characterized by nanoparticle tracking analysis, transmission electron microscope, and western blotting. Their morphology and protein composition are similar to native EVs, indicating that EMs may potentially serve as a low-cost and high-yield surrogate of native EVs for clinical translations.

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, ...