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

엔도솜 유래 나노소포의 최적화된 자기 고수율 생산

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.

저자 스포트라이트: 자성 나노입자를 이용한 엑소좀 모방을 위한 대규모의 재현 가능한 생산 방법 개발 

엔도솜 유래 소포의 대규모 생산을 위한 자기 분리 보조 고속 균질화 방법

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  

배양된 세포에서 자성 나노입자를 함유한 엔도솜의 분리 및 준비

먼저 6웰 플레이트에서 10% FBS와 5% 페니실린-스트렙토마이신을 함유한 DMEM Complete Medium에서 세포를 배양합니다. 섭씨 37도, 이산화탄소 5%에서 하룻밤 동안 배양액을 배양합니다. 다음날 세포를 10 나노 몰 폴리 리신 변성 산화철 나노 입자 또는 IONP와 공동 배양하고 섭씨 37도에서 12 시간 동안 5 % 이산화탄소를 배양합니다.배양 후 웰당 0.5mL의 트립신을 3분 동안 첨가하여 세포를 소화합니다. 그런 다음 웰당 1m리터의 Complete Medium을 추가하여 소화를 중지시킵니다. 세포 현탁액을 1, 5 분 동안 000 g에 분리하고, PBS에 있는 펠릿을 다시 현탁시키기 전에 상청액을 폐기하십시오.그런 다음 펠릿을 8ml의 갓 준비된 저긴장성 용액에 15분 동안 다시 현탁시킵니다. 유리제 시험관에 세포 현탁액을 옮기고, 유리제 균질화기를 사용하여, 1, 000 분당 회전수에 세포 기관을 풀어 놓기 위하여 20의 충격을 적용하십시오. 균질화 후 세포 현탁액 1mL를 1.5mL 미세 원심분리 튜브로 옮깁니다.튜브를 자기 분리기에 1시간 동안 넣어 IONP가 장착된 엔도솜을 다른 세포 기관 및 세포 파편과 완전히 분리합니다. 엔도솜을 수집하려면 튜브에서 액체를 버리고 3ml의 PBS를 추가하여 엔도솜을 다시 부유시킵니다. 그런 다음 피펫 건을 사용하여 마그네틱 프레임 옆에 있는 튜브의 접촉면에 있는 갈색 펠릿을 다시 매달기 위해 불어냅니다.고속 균질화를 위해 엔도좀 용액을 15밀리리터 원심분리기 튜브로 옮깁니다. 아이스박스를 튜브 아래에 놓은 다음 튜브를 고속 균질화기에 놓습니다. 바닥을 건드리지 않고 10밀리리터 프로브를 튜브에 넣습니다.균질화기 화면에서 속도를 140g으로 조정하고 시간을 5분으로 설정합니다. OK 버튼을 누른 다음 시작 버튼을 누릅니다. 고속 균질화 후 나노 소포 현탁액을 1.5 밀리리터 튜브로 옮기고 튜브를 자기 분리기에 1 시간 동안 놓습니다.마지막으로, 마그네틱 프레임 옆의 표면을 방해하지 않고 필요한 엑소좀 모방체 또는 EM이 포함된 액체를 수집합니다. NTA와 TEM에 의해 분석된 BMSC-EM의 형태는 지질 이중층으로 구분되는 전형적인 그릇 모양의 소포 같은 구조를 보여주었습니다. BMSC-EM과 293T-EM은 모두 기본 EV와 유사한 유체역학적 직경을 가졌습니다.웨스턴 블로팅 결과에 따르면 BMSC-EM은 EV와 동일한 단백질 바이오마커를 함유하고 있으며 원형질막 오염이 거의 없는 것으로 나타났습니다. EMS의 수율은 초원심분리로 제조된 네이티브 EV보다 훨씬 높았습니다. EM은 네이티브 EV와 유사한 단백질 구성을 가지고 있었습니다.

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, ...