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

Produzione magnetica ad alto rendimento ottimizzata di nanovescicole derivate da endosomi

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.

Autore in primo piano: Sviluppo di un metodo di produzione riproducibile su larga scala per mimetici esosomici utilizzando nanoparticelle magnetiche 

Un metodo di omogeneizzazione ad alta velocità assistito da separazione magnetica per la produzione su larga scala di vescicole derivate da endosomi

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  

Isolamento e preparazione di endosomi contenenti nanoparticelle magnetiche da cellule in coltura

Per iniziare, coltivare le cellule in DMEM Complete Medium contenente il 10% di FBS e il 5% di penicillina-streptomicina in una piastra a sei pozzetti. Incubare la coltura per una notte a 37 gradi Celsius e al 5% di anidride carbonica. Il giorno successivo, co-coltivare le cellule con 10 nanoparticelle nanomolari di ossido di ferro modificato con polilisina o IONP e incubare a 37 gradi Celsius e al 5% di anidride carbonica per 12 ore.Dopo l'incubazione, aggiungere 0,5 millilitri di tripsina per pozzetto per tre minuti per digerire le cellule. Quindi aggiungere un millilitro di Complete Medium per pozzetto per fermare la digestione. Centrifugare la sospensione cellulare a 1.000 g per cinque minuti ed eliminare il surnatante prima di risospendere il pellet in PBS.Quindi sospendere nuovamente il pellet in otto millilitri di soluzione ipotonica appena preparata per 15 minuti. Trasferire la sospensione cellulare in una provetta di vetro e, utilizzando un omogeneizzatore di vetro, applicare 20 scosse a 1.000 giri/min per rilasciare gli organelli. Dopo l'omogeneizzazione, trasferire un millilitro della sospensione cellulare in una provetta per microcentrifuga da 1,5 millilitri.Posizionare la provetta in un separatore magnetico per un'ora per separare completamente gli endosomi caricati con IONP da altri organelli e detriti cellulari. Per raccogliere gli endosomi, scartare il liquido dalla provetta e aggiungere tre millilitri di PBS per risospendere gli endosomi. Quindi utilizzare una pistola a pipetta per soffiare per risospendere i pellet marroni che si trovano sulla superficie di contatto del tubo accanto al telaio magnetico.Per l'omogeneizzazione ad alta velocità, trasferire la soluzione endosomica in una provetta da centrifuga da 15 millilitri. Posizionare la ghiacciaia sotto il tubo, quindi posizionare il tubo sull'omogeneizzatore ad alta velocità. Posizionare la sonda da 10 millilitri nel tubo senza toccare il fondo.Sullo schermo dell'omogeneizzatore, regolare la velocità a 140 g e impostare il tempo per cinque minuti. Premere il pulsante OK seguito dal pulsante Start. Dopo l'omogeneizzazione ad alta velocità, trasferire la sospensione di nano-vescicole in una provetta da 1,5 millilitri e posizionare la provetta in un separatore magnetico per un'ora.Infine, raccogli il liquido contenente i mimici esosomi o EM richiesti senza disturbare la superficie accanto al telaio magnetico. La morfologia dei BMSC-EM analizzati mediante NTA e TEM ha mostrato una tipica struttura vescicolare a forma di ciotola, delimitata da un doppio strato lipidico. Sia i BMSC-EM che i 293T-EM avevano un diametro idrodinamico simile a quello degli EV nativi.I risultati del Western blotting hanno mostrato che le BMSC-EM contengono gli stessi biomarcatori proteici delle vescicole extracellulari e non presentano quasi alcuna contaminazione della membrana plasmatica. La resa dell'EMS è risultata significativamente superiore rispetto alle vescicole extracellulari native preparate mediante ultracentrifugazione. Gli EM avevano una composizione proteica simile a quella delle EV native.

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, ...