Name: Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells
Uploaded: 2024-01-26T14:10:00
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isolation and preparation of endosomes containing magnetic nanoparticles from cultured cells

优化内体衍生的纳米囊泡的磁性高产率生产

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.

作者聚焦：使用磁性纳米颗粒开发大规模、可重现的外泌体模拟物生产方法 

一种用于大规模生产内体衍生囊泡的磁分离辅助高速均质化方法

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  

从培养细胞中分离和制备含有磁性纳米颗粒的内体

首先，在含有 10% FBS 和 5% 青霉素 - 链霉素的 DMEM 完全培养基中，在六孔板中培养细胞。将培养物在 37 摄氏度和 5% 二氧化碳下孵育一夜。第二天，将细胞与 10 纳摩尔聚赖氨酸修饰的氧化铁纳米颗粒或 IONP 共培养，并在 37 摄氏度和 5% 二氧化碳下孵育 12 小时。孵育后，每孔加入 0.5 毫升胰蛋白酶 3 分钟以消化细胞。然后每孔加入一毫升完全培养基以停止消化。将细胞悬浮液以 1， 000 g 离心 5 分钟，并弃去上清液，然后将沉淀重新悬浮在 PBS 中。然后将沉淀重新悬浮在8毫升新鲜制备的低渗溶液中15分钟。将细胞悬浮液转移到玻璃试管中，并使用玻璃均质器，以1， 000 RPM的速度施加20次冲击以释放细胞器。均质化后，将一毫升细胞悬浮液转移到1.5毫升微量离心管中。将试管置于磁性分离器中一小时，以将负载 IONP 的内体与其他细胞器和细胞碎片完全分离。要收集内体，请从试管中丢弃液体，并加入三毫升 PBS 以重新悬浮内体。然后使用移液枪吹气，以重新悬浮在磁架旁边的管子接触面上的棕色颗粒。对于高速均质化，将内体溶液转移到 15 毫升离心管中。将冰箱放在管子下面，然后将管子放在高速均质机上。将 10 毫升探头放入管中，不要接触底部。在均质机屏幕上，将速度调整为140g，并将时间设置为五分钟。按 OK 按钮，然后按开始按钮。高速均质化后，将纳米囊泡悬浮液转移到1.5毫升试管中，并将试管置于磁选机中1小时。最后，收集含有所需外泌体模拟物或电磁镜的液体，而不会干扰磁性框架旁边的表面。NTA和TEM分析的BMSC-EMs形态呈典型的碗状囊泡状结构，由脂质双层分隔。BMSC-EM 和 293T-EM 都具有与原生 EV 相似的流体动力学直径。Western blotting结果显示，BMSC-EMs含有与EVs相同的蛋白质生物标志物，并且几乎没有质膜污染。EMS的收率显著高于超速离心制备的天然EV。EM具有与天然EV相似的蛋白质组成。

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, ...