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

Summary

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).

Abstract

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.

Introduction

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 modulation^3,4. 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 EVs^6,7,8. 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 sectioning^9,10, demonstrating potent biological functions as native EVs^11,12. 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 patients^15,16. 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.

Protocol

NOTE: A schematic of the method is shown in Figure 1.

1. EM preparation and isolation

1. Cell internalization of MNPs
   1. Cell culture
      1. Suspend 1 × 10⁶ rat bone marrow mesenchymal stem cells (BMSC), 293T cells, or Mouse ovarian epithelial cancer cells (ID8) in DMEM complete medium with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin solution (P/S) in 2 mL per six-well plate (see Table of Materials).
      2. Culture the cells overnight at 37 °C and 5% CO₂.
         NOTE: The number of cells after adherence was about 1 × 10⁶.
   2. Endocytosis of MNPs
      1. Reduce the medium volume of each six-well plate to 1 mL. Co-culture the cells with 10 nm polylysine-modified iron oxide nanoparticles (IONPs) (see Table of Materials) at a concentration of 100 µg/1 × 10⁶ cells and incubate at 37 °C in an atmosphere containing 5% CO₂ for 12 h to allow the cells to endocytose IONPs fully.
2. Isolation and homogenization of endosomes
   1. Cell hypotonic treatment
      1. Digest the cells that have fully internalized IONPs with 0.5 mL/well trypsin for 3 min, and terminate the digestion by adding 1 mL/well complete medium.
      2. Centrifuge at 1000 x g for 5 min, and discard the supernatant. Resuspend the cells in phosphate buffer saline (PBS) and centrifuge, repeatedly twice to wash out the residual medium and unabsorbed IONPs.
      3. Finally, resuspend the pellet in 8 mL in pre-prepared hypotonic solution (71.4 mM potassium chloride, 1.3 mM sodium citrate, pH 7.2-7.4) for 15 min (see Table of Materials).
   2. Release of organelles by low-speed homogenization
      1. Transfer the cell suspension in hypotonic solution to a glass test tube and release the organelles using a glass homogenizer (see Table of Materials) by 20 shocks at 1000 rpm.
   3. Magnetic separation to obtain endosomes
      1. Transfer 1 mL of the homogenized cell solution to a 1.5 mL microcentrifuge tube and place in a magnetic separator for 1 h to fully separate IONP-loaded endosomes from other organelles (such as nucleus and mitochondria) and cell debris.
      2. Collect the brown pellets on the contact surface of the microcentrifuge tubes next to the magnetic frame (see Table of Materials), i.e., the IONP-loaded endosomes. Discard the liquid in the microcentrifuge tubes and add 3 mL of PBS to resuspend the endosomes.
3. High-speed homogenization
   1. Transfer the solution containing the endosomes to a 15 mL centrifuge tube with a liquid volume between 3-10 mL.
   2. Extend the 10 mm probe of the high-speed homogenizer (see Table of Materials) to the bottom of the centrifuge tube without touching the bottom. Adjust the Speed button under the screen, set the speed to 140 x g, and adjust the Time button to set the time of 5 min, then press the OK button.
   3. Press the Start button to carry out the homogenization after placing an ice box under the sample.
4. Magnetic sorting for EMs
   1. Transfer the solution containing nanovesicles obtained from high-speed homogenization into the 1.5 mL microcentrifuge tube and put it in a magnetic separator for 1 h.
   2. Collect the liquid that contains the required EMs. Be careful not to touch the surface of the tubes next to the magnetic frame, which adsorbed free IONPs and IONP-loaded EMs.

2. EM characterization (Figure 2 and Figure 3)

1. Dynamic light scattering (DLS) analysis
   1. Inject 1 mL of EMs sample (20 µg/mL) along the wall into the cuvette and place it in the instrument sample slot of the DLS instrument (see Table of Materials).
   2. Open the DLS software, select Particle Size Test, and start testing.
   3. Measure the protein concentration using a BCA protein assay kit (see Table of Materials).
2. Nanoparticle tracking analysis (NTA)
   1. Dilute EMs with PBS to a solution at 1 × 10⁷-1 × 10⁸ particles/mL and inject them into the chamber of the NTA instrument (see Table of Materials) using a 1 mL syringe at a flow rate of 500-1000 µL/min.
   2. Open the 488 laser channels and ensure the number of EMs in the software interface is within 100-300 and in Brownian motion.
   3. According to the manufacturer's protocol, select the appropriate SOP (EV-488) to ensure the instrument's sensitivity is suitable for EM detection.
3. Transmission electron microscopy (TEM)
   1. Fix EMs (1 mg/mL) with an equal volume of 5% glutaraldehyde for 30 min, and quantify the concentration of EMs as 1 mg/mL by BCA protein assay kit.
   2. Place a 10 µL drop of EMs on the carbon side of the copper mesh, and remove excess liquid from the side with filter paper after 10 min. Add 10 µL of PBS and blot immediately with filter paper. Repeat twice to remove excess glutaraldehyde.
   3. Drop 5 µL of uranyl acetate contrast agent and negatively stain for 1 min, then remove the excess contrast agent. Wash three times with deionized water and dry at room temperature (RT).
   4. Record image by a TEM at an acceleration voltage of 100 kV.
4. Western blotting
   1. Add 100 µL of cell lysis buffer and 5 µL of a 50x protease inhibitors cocktail to the samples (see Table of Materials). Mix gently with a pipette gun and put on ice for 30 min.
   2. Centrifuge the solution at 1000 x g for 10 min at 4 °C, quantify the protein concentration of the supernatant with a BCA protein assay kit, and collect it.
   3. Mix the 100 µL supernatant with 25 µL of 5x protein loading buffer and heat at 95 °C for 10 min.
   4. Prepare gels according to the instructions of the preformed gels. Load 15 µg of protein per well and run by gel electrophoresis at 80 V. Wait for the sample to run to the separation gel, change to 100 V, and then transfer the protein to a nitrocellulose membrane at 100 V, 60 min under ice bath.
   5. Detect the non-EV-specific markers (Calnexin) and EV biomarkers (Annexin and CD63) by western blotting (see Table of Materials).
      ​NOTE: The antibodies are diluted as per the manufacturer's recommendations.

3. In vitro EM function detection

1. EMs labeling
   1. Mix 1 µL of PKH26 with 250 µL of diluent C (1:250) to make 2x staining solution (4 × 10^-6 M) (see Table of Materials).
   2. Mix 250 µL of EMs (1 mg/mL) with 250 µL of 2x staining solution and quickly blow with a pipetting gun.
   3. Incubate at RT for 2-5 min while gently inverting the centrifuge tube.
   4. Add an equal volume of serum or 1% bovine serum protein and incubate for 1 min to terminate the digestion.
   5. Remove excess PKH26 by ultrafiltration with 1000 KD ultrafiltration tubes at 3000 x g for 30 min, and repeat three times by adding PBS. Resuspend the remaining liquid to 500 µL with PBS and filter through a 0.22 µm filter.
2. Cells uptake assay
   1. Cell inoculation: Seed 1 × 10⁶ cells in 35 mm confocal dishes with 1 mL of DMEM containing 10% FBS and 5% P/S, and incubate at 37 °C and 5% CO₂ overnight.
   2. Filter the EMs with 0.22 µm sterile syringe filters to remove potential contamination.
   3. Treat the cells with PKH26-labeled EMs (10⁹ particles/mL) for 8 h.
   4. Wash three times with PBS, add DAPI (10 ng/mL), and incubate for 10 min at RT.
   5. Acquire the fluorescence images in confocal laser scanning fluorescence microscopy using the 405 nm and 561 nm laser channels (see Table of Materials).

Results

The workflow of EM preparation by magnetic separation-assisted high-speed homogenization is shown in Figure 1. Cells internalize 10 nm polylysine-modified IONPs, which are specifically accumulated in endosomes via endocytosis (Figure 3A). After being treated with hypotonic buffer and homogenized, the IONP-loaded endosomes are released from the cells and subsequently collected by magnetic separation. The isolated endosomes are further reconstituted into monodisperse nanovesicles, also known as EMs, by high-speed homogenization. We explored multiple key parameters (e.g., homogenization speed and time) to identify optimized EM preparation conditions (Figure 2). Finally, a homogenization speed of 140 x g for 5 min was chosen as the optimized condition by considering the particle size and yield of produced EMs. Free IONPs and IONP-loaded EMs are eventually removed from the final product solution by a second round of magnetic separation to obtain IONP-free EMs. The method produces highly uniform and monodispersed nanovesicles from parental cell endosomes, sharing the same biological origin as native EVs.

To compare EVs obtained by ultracentrifugation with EMs generated by this method, BMSC and 293T were prepared for EVs and EMs. The diameter and morphology of EMs were analyzed by NTA and TEM. The morphology of BMSC-EMs has the feature of a typical bowl-shaped vesicle-like structure and is delimited by a lipid bilayer (Figure 3A). As analyzed by NTA, both BMSC-EMs and 293T-EMs have a similar hydrodynamic diameter to native EVs (BMSC-EVs and 293T-EVs) (Figure 3B). The BMSC-EMs yields of high-speed homogenization were 8.16 × 10⁸-1.42 × 10⁹/1 × 10⁶cells, and the yield of native EVs prepared by ultracentrifugation was only 7.2 × 10⁷-1.12 × 10⁸/1 × 10⁶cells. Similarly, the 293T-EMs yields of high-speed homogenization were 3.71 × 10⁸-7.58 × 10⁸/1 × 10⁶cells, which reaches up to approximately 100-fold higher than those of native 293T-EVs prepared by the conventional ultracentrifuge method (≈5.5 × 10⁶/1 × 10⁶ cells) (Figure 4A).

Moreover, western blotting results showed that BMSC-EMs contain the same protein biomarkers as EVs (CD63 and Annexin). Both EMs and EVs are negative for Calnexin expression, suggesting that EMs produced by this method had almost no plasma membrane contamination (Figure 3C). There is no significant difference in protein concentration between EM and EV, BMSC-EMs and BMSC-EVs exhibited similar total protein concentrations, 11.15 µg/1 × 10⁹ particles and 14.71 µg/1 × 10⁹ particles via the BCA protein assay kit. Moreover, 293T-EMs and 293T-EVs exhibited total protein concentrations of approximately 31.8 µg/1 × 10⁹ particles and 9.95 µg/1 × 10⁹ particles, respectively (Figure 4B). These results indicate that EMs have a similar protein composition as native EVs. To detect whether EMs can be endocytosed, PKH26-labeled EMs and EVs were co-incubated with BMSC and ID8 at a concentration of 1 × 10⁹ particles/mL for 8 h, and the cells were observed under confocal fluorescence microscopy to confirm that EMs could be readily taken up by the cells for action as well as EVs (Figure 5).

Figure 1: Schematic diagram of the magnetic-assisted high-speed homogenization method. Step 1: Cells internalize IONPs into endosomes through endocytosis. Step 2: Collect organelles, including IONP-loaded endosomes, after hypotonic treatment and homogenization. Step 3: Purify IONP-loaded endosomes by magnetic separation. Step 4: The endosomes are homogenized and reconstituted into monodisperse nanovesicles. Step 5: Remove free IONPs and IONP-loaded EMs by magnetic separation to collect IONP-free EMs. Please click here to view a larger version of this figure.

Figure 2: The optimization of EM preparation conditions. (A) The diameter and PDI of BMSC-EMs in response to homogenization speed changes when time is set at 5 min were analyzed by DLS. (B) The diameter and PDI of BMSC-EMs in response to homogenization time changes when the homogenization speed is set at 140 x g were analyzed by DLS. (C) The diameter and PDI of BMSC-EMs at different storage time points were analyzed by DLS. (D) The diameter and concentration of BMSC-EMs in response to homogenization speed changes when time is set at 5 min were analyzed by NTA. (E) The diameter and concentration of BMSC-EMs in response to the homogenization time change when the homogenization speed is set at 140 x g were analyzed by NTA. (F) The diameter and yield of BMSC-EMs in response to BMSC cells co-incubation with IONPs of different concentrations were analyzed by NTA. ****p < 0.0001. Please click here to view a larger version of this figure.

Figure 3: Characterization of EMs. (A) TEM characterization of the morphology of parental cells with endocytosed IONPs, endosomes, and EMs. The scale bars represent 500 nm. (B) Hydrodynamic diameters of BMSC-EM, BMSC-EV, 293T-EM, and 293T-EV are characterized by NTA. (C) Western blot analysis results of BMSC-EMs, BMSC-EVs, and BMSC cell lysates. Please click here to view a larger version of this figure.

Figure 4: The yield of EMs is higher than EVs. (A) The yield of EMs and EVs prepared from BMSC and 293T was analyzed by NTA. (B) Protein yield of EMs and EVs prepared from BMSC and 293T. **p < 0.01. Please click here to view a larger version of this figure.

Figure 5: Representative fluorescent microscope images of PKH26-labeled EVs and EMs internalized by BMSC and ID8. The scale bars represent 10 µm. Please click here to view a larger version of this figure.

Discussion

As a surrogate of cell-free therapy and a nanoscale drug delivery system, EVs have yet to meet their clinical expectations, and a main obstacle is the lack of scalable and reproducible production and purification methods⁶. Therefore, various types of EMs have been developed as EV analogs with similar biological complexity¹⁴. To date, the most commonly used EM example is cell plasma membrane-derived nanovesicles. The preparation of such nanovesicles is relatively easy and straightforward by directly extruding whole parent cells¹⁷. However, cell plasma membrane-derived nanovesicles cannot recapitulate native EVs owing to two drawbacks: First, the biological origin of these nanovesicles is from cell plasma membrane, which contains different lipid and protein compositions in comparison with native EVs; Second, the contaminations, including proteins, nucleic acids and lipids from non-EV organelles and cell debris, causing inevitable EM heterogeneity. In this method, we incubated cells with IONPs and collected IONP-loaded endosomes through magnetic separation, then generated monodisperse nanovesicles via high-speed homogenization, eventually obtaining IONP-free EMs by a second round of magnetic separation. Moreover, we optimized the EM preparation conditions by exploring the impact of different parameters, such as homogenization speed and time, on EM size and yield. This protocol takes advantage of an interesting biological phenomenon: MNPs (e.g., 10 nm IONPs) can be efficiently internalized by almost all cells via endocytosis and exclusively accumulated in endosomes, not other organelles. This unique biological process enabled the isolation of MNP-loaded endosomes without non-EV contaminations via magnetic separation. In return, nanovesicles prepared by these purified cell endosomes could more faithfully recapitulate the biological complexity of native EVs.

Moreover, the golden standard of the EV isolation method is ultracentrifugation, which costs massive cell culture media and a long processing time of over 5 h and only produces 1 × 10⁷-1 × 10⁸ particles from 1 x 10⁶ cells⁶. Traditional whole parental cell extrusion methods include multiple steps involving ultra-high pressure membrane extrusion and ultracentrifugation, which have a relatively higher yield but require expensive equipment¹⁷. However, the EM production yield, efficiency, cost, and manpower are substantially improved by utilizing our protocol. The high-speed homogenizer is a common and low-cost equipment for a short processing time of 5 min in this protocol, and this method can produce up to 100 times more EMs from 1 × 10⁶ cells in comparison with native EVs. However, this method has some limitations, such as loss of endosomal proteins during high-speed homogenization, and it has been shown that MNPs endocytosed by cells can be transferred to lysosomes, which would be a potential contamination¹⁸.

In perspective, EMs can be engineered to exert important biological functions as their native counterparts, which may serve as a novel and promising type of biological therapeutics. Bioactive substances (e.g., proteins and nucleic acids) can be integrated into EMs via selecting specific parental cells (e.g., stem cells¹⁹) or genetic modification (e.g., fusion proteins²⁰). For example, cell-derived nanovesicles by extruding living embryonic stem cells have a positive effect on the recovery or wound-healing process¹⁹. Genetic modification is an alternative approach to generate EMs with specific biological functions. For example, when cells were transfected with vectors of anti-epidermal growth factor receptor (EGFR) nanobodies fused to glycosylphosphatidylinositol (GPI) anchor signal peptides, the EGFR nanobodies can be anchored on the surface of EVs for targeting EGFR-expressing tumor cells²⁰. EMs have performed well as a surrogate of cell-free therapy and a nanoscale drug delivery system in multiple preclinical studies¹⁴, but there is a lack of a comprehensive mechanistic understanding of EM or EV-based therapy with concerns about the heterogeneity and reproducibility of EVs and EMs in clinic investigations. Taken together, the biological functions of EMs and their clinical implications warrant further investigations.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin antibody	ABclonal	A11235	Western blotting
BCA assay kit	Beyotime	P0012	Protein concentration assay
Calnexin	GeneTex	HL1598	Western blotting
CD63 antibody	ABclonal	A19023	Western blotting
Cell lysis buffer for Western and IP	Beyotime	P0013	Western blotting
Centrifuge	Beckman	Allegra X-30R	Cell centrifuge
CO2 incubator	Thermo		Cell culture
Confocal laser scanning fluorescence microscopy	NIKON	A1 HD25	Photo the fluorescence picture
DMEM basic (1x)	GIBCO	C11995500BT	Cell culture
Dynamic light scattering (DLS)	Malvern	Zetasizer Nano ZS ZEN3600	Diameter analysis
Electric glass homogenizer	SCIENTZ(Ningbo, China)	DY89-II	Low-speed homogenization
Exosome-depleted FBS	system Bioscience	EXO-FBS-50A-1	Cell culture
High-speed homogenizer	SCIENTZ(Ningbo, China)	XHF-DY	High-speed homogenization
Magnetic grate	Tuohe Electromechanical Technology (Shanghai, China)	NA	Magnetic separation
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling	Sigma-Aldrich	PKH26GL-1KT	The kit contains PKH26 cell linker in ethanol and Diluent C
Polylysine-modified iron oxide nanoparticles (IONPs)	Zhongke Leiming Technology (Beijing, China)	Mag1100-10	Cell culture
Potassium chloride	Aladdin	7447-40-7	Cell hypotonic treatment
Protease inhibitor cocktail	Beyotime	P1030	Proteinase inhibitor
Sodium citrate	Aladdin	7447-40-7	Cell hypotonic treatment
Transmission electron microscopy (TEM)	JEOL	JEM-2100plus	Morphology image
Ultracentrifuge	Beckman	Optima XPN-100	Exosome centrifuge
ZetaView nanoparticle  tracking analyzers	Particle Metrix	PMX120	Nanoparticle tracking analysis