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

Produção magnética otimizada de alto rendimento de nanovesículas derivadas de endossomas

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.

Autor em destaque: Desenvolvimento de um método de produção reprodutível em larga escala para miméticos de exossomos usando nanopartículas magnéticas 

Um Método de Homogeneização de Alta Velocidade Assistida por Separação Magnética para Produção em Larga Escala de Vesículas Derivadas do Endossomo

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 Preparação de Endossomos Contendo Nanopartículas Magnéticas a partir de Células Cultivadas

Para começar, cultivar as células em meio completo DMEM contendo 10% FBS e 5% penicilina-estreptomicina em uma placa de seis poços. Incubar a cultura durante uma noite a 37 graus Celsius e 5% de dióxido de carbono. No dia seguinte, co-cultivar as células com 10 nanomolares nanomolares de polilisina modificada por óxido de ferro ou IONPs, e incubar a 37 graus Celsius e 5% de dióxido de carbono por 12 horas.Após a incubação, adicione 0,5 mililitros de tripsina por poço por três minutos para digerir as células. Em seguida, adicione um mililitro de Meio Completo por poço para interromper a digestão. Centrifugar a suspensão celular a 1.000 g durante cinco minutos e eliminar o sobrenadante antes de voltar a suspender o pellet em PBS.Em seguida, ressuspenda o pellet em oito mililitros de solução hipotônica recém-preparada por 15 minutos. Transfira a suspensão celular para um tubo de ensaio de vidro e, usando um homogeneizador de vidro, aplique 20 choques a 1.000 RPM para liberar as organelas. Após homogeneização, transferir um mililitro da suspensão celular para um tubo de microcentrífuga de 1,5 mililitro.Coloque o tubo em um separador magnético por uma hora para separar completamente os endossomos carregados com IONP de outras organelas e detritos celulares. Para coletar endossomos, descarte o líquido do tubo e adicione três mililitros de PBS para ressuspender os endossomos. Em seguida, use uma pistola de pipeta para soprar novamente para ressuspender as pelotas marrons encontradas na superfície de contato do tubo ao lado da estrutura magnética.Para homogeneização de alta velocidade, transfira a solução endossomal para um tubo centrífugo de 15 mililitros. Coloque a caixa de gelo sob o tubo e, em seguida, coloque o tubo no homogeneizador de alta velocidade. Coloque a sonda de 10 mililitros no tubo sem tocar no fundo.Na tela do homogeneizador, ajuste a velocidade para 140 g e defina o tempo para cinco minutos. Pressione o botão OK seguido pelo botão Iniciar. Após homogeneização em alta velocidade, transfira a suspensão da nanovesícula para um tubo de 1,5 mililitro e coloque o tubo em um separador magnético por uma hora.Finalmente, colete o líquido contendo as imitações ou EMs do exossomo necessários sem perturbar a superfície próxima ao quadro magnético. A morfologia das BMSC-MEs analisadas por NTA e MET mostrou uma estrutura vesícula típica em forma de tigela, delimitada por uma bicamada lipídica. Tanto os BMSC-MEs quanto os 293T-EMs apresentaram diâmetro hidrodinâmico semelhante aos EVs nativos.Os resultados de Western blotting mostraram que os BMSC-EMs contêm os mesmos biomarcadores proteicos que os EVs, e quase não tiveram contaminação da membrana plasmática. O rendimento de EMS foi significativamente maior do que os EVs nativos preparados por ultracentrifugação. Os MEs apresentaram composição proteica semelhante aos EVs nativos.

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, ...