The authors acknowledge the use of instruments at the Shared Instrumentation Core Facility at the Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences. This study was supported by the National Natural Science Foundation of China (NSFC; 82172598), the Natural Science Foundation of Zhejiang Province, China (LZ22H310001), the 551 Health Talent Training Project of Health Commission of Zhejiang Province, China, the Agricultural and Social Development Research Project of Hangzhou Municipal Science and Technology Bureau (2022ZDSJ0474) and Qiantang Interdisciplinary Research Grant.

NOTE: A schematic of the method is shown in Figure 1.
1. EM preparation and isolation
<ol>
	<li>Cell internalization of MNPs
	<ol>
		<li>Cell culture
		<ol>
			<li>Suspend 1 &#215; 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).</li>
			<li>Culture the cells overnight at 37 &#176;C and 5% CO2. 
			NOTE: The number of cells after adherence was about 1 &#215; 106.</li>
		</ol>
		</li>
		<li>Endocytosis of MNPs
		<ol>
			<li>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 &#181;g/1 &#215; 106 cells and incubate at 37 &#176;C in an atmosphere containing 5% CO2 for 12 h to allow the cells to endocytose IONPs fully.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolation and homogenization of endosomes
	<ol>
		<li>Cell hypotonic treatment
		<ol>
			<li>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.</li>
			<li>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.</li>
			<li>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).</li>
		</ol>
		</li>
		<li>Release of organelles by low-speed homogenization
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Magnetic separation to obtain endosomes
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>High-speed homogenization
	<ol>
		<li>Transfer the solution containing the endosomes to a 15 mL centrifuge tube with a liquid volume between 3-10 mL.</li>
		<li>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.</li>
		<li>Press the Start button to carry out the homogenization after placing an ice box under the sample.</li>
	</ol>
	</li>
	<li>Magnetic sorting for EMs
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. EM characterization (Figure 2 and Figure 3)
<ol>
	<li>Dynamic light scattering (DLS) analysis
	<ol>
		<li>Inject 1 mL of EMs sample (20 &#181;g/mL) along the wall into the cuvette and place it in the instrument sample slot of the DLS instrument (see Table of Materials).</li>
		<li>Open the DLS software, select Particle Size Test, and start testing.</li>
		<li>Measure the protein concentration using a BCA protein assay kit (see Table of Materials).</li>
	</ol>
	</li>
	<li>Nanoparticle tracking analysis (NTA)
	<ol>
		<li>Dilute EMs with PBS to a solution at 1 &#215; 107-1 &#215; 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 &#181;L/min.</li>
		<li>Open the 488 laser channels and ensure the number of EMs in the software interface is within 100-300 and in Brownian motion.</li>
		<li>According to the manufacturer&#39;s protocol, select the appropriate SOP (EV-488) to ensure the instrument&#39;s sensitivity is suitable for EM detection.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>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.</li>
		<li>Place a 10 &#181;L 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 &#181;L of PBS and blot immediately with filter paper. Repeat twice to remove excess glutaraldehyde.</li>
		<li>Drop 5 &#181;L 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).</li>
		<li>Record image by a TEM at an acceleration voltage of 100 kV.</li>
	</ol>
	</li>
	<li>Western blotting
	<ol>
		<li>Add 100 &#181;L of cell lysis buffer and 5 &#181;L 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.</li>
		<li>Centrifuge the solution at 1000 x g for 10 min at 4 &#176;C, quantify the protein concentration of the supernatant with a BCA protein assay kit, and collect it.</li>
		<li>Mix the 100 &#181;L supernatant with 25 &#181;L of 5x protein loading buffer and heat at 95 &#176;C for 10 min.</li>
		<li>Prepare gels according to the instructions of the preformed gels. Load 15 &#181;g 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.</li>
		<li>Detect the non-EV-specific markers (Calnexin) and EV biomarkers (Annexin and CD63) by western blotting (see Table of Materials). 
		&#8203;NOTE: The antibodies are diluted as per the manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
</ol>
3. In vitro EM function detection
<ol>
	<li>EMs labeling
	<ol>
		<li>Mix 1 &#181;L of PKH26 with 250 &#181;L of diluent C (1:250) to make 2x staining solution (4 &#215; 10-6 M) (see Table of Materials).</li>
		<li>Mix 250 &#181;L of EMs (1 mg/mL) with 250 &#181;L of 2x staining solution and quickly blow with a pipetting gun.</li>
		<li>Incubate at RT for 2-5 min while gently inverting the centrifuge tube.</li>
		<li>Add an equal volume of serum or 1% bovine serum protein and incubate for 1 min to terminate the digestion.</li>
		<li>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 &#181;L with PBS and filter through a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Cells uptake assay
	<ol>
		<li>Cell inoculation: Seed 1 &#215; 106 cells in 35 mm confocal dishes with 1 mL of DMEM containing 10% FBS and 5% P/S, and incubate at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Filter the EMs with 0.22 &#181;m sterile syringe filters to remove potential contamination.</li>
		<li>Treat the cells with PKH26-labeled EMs (109 particles/mL) for 8 h.</li>
		<li>Wash three times with PBS, add DAPI (10 ng/mL), and incubate for 10 min at RT.</li>
		<li>Acquire the fluorescence images in confocal laser scanning fluorescence microscopy using the 405 nm and 561 nm laser channels (see Table of Materials).</li>
	</ol>
	</li>
</ol>

Optimized Magnetic High-Yield Production of Endosome-Derived Nanovesicles

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D.W. and P.G. are co-inventors of a patent application filed by the Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences. The other author declares no conflicts of interest.

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 st...

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.

<table><tbody><tr><td>Annexin antibody</td><td>ABclonal</td><td>A11235</td><td>Western blotting</td></tr><tr><td>BCA assay kit</td><td>Beyotime</td><td>P0012</td><td>Protein concentration assay</td></tr><tr><td>Calnexin</td><td>GeneTex</td><td>HL1598</td><td>Western blotting</td></tr><tr><td>CD63 antibody</td><td>ABclonal</td><td>A19023</td><td>Western blotting</td></tr><tr><td>Cell lysis buffer for Western and IP</td><td>Beyotime</td><td>P0013</td><td>Western blotting</td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>Allegra X-30R</td><td>Cell centrifuge</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo</td><td></td><td>Cell culture</td></tr><tr><td>Confocal laser scanning fluorescence microscopy</td><td>NIKON</td><td>A1 HD25</td><td>Photo the fluorescence picture</td></tr><tr><td>DMEM basic (1x)</td><td>GIBCO</td><td>C11995500BT</td><td>Cell culture</td></tr><tr><td>Dynamic light scattering (DLS)</td><td>Malvern</td><td>Zetasizer Nano ZS ZEN3600</td><td>Diameter analysis</td></tr><tr><td>Electric glass homogenizer</td><td>SCIENTZ(Ningbo, China)</td><td>DY89-II</td><td>Low-speed homogenization</td></tr><tr><td>Exosome-depleted FBS</td><td>system Bioscience</td><td>EXO-FBS-50A-1</td><td>Cell culture</td></tr><tr><td>High-speed homogenizer</td><td>SCIENTZ(Ningbo, China)</td><td>XHF-DY</td><td>High-speed homogenization</td></tr><tr><td>Magnetic grate</td><td>Tuohe Electromechanical Technology (Shanghai, China)</td><td>NA</td><td>Magnetic separation</td></tr><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma-Aldrich</td><td>PKH26GL-1KT</td><td>The kit contains PKH26 cell linker in ethanol and Diluent C</td></tr><tr><td>Polylysine-modified iron oxide nanoparticles (IONPs)</td><td>Zhongke Leiming Technology (Beijing, China)</td><td>Mag1100-10</td><td>Cell culture</td></tr><tr><td>Potassium chloride</td><td>Aladdin</td><td>7447-40-7</td><td>Cell hypotonic treatment</td></tr><tr><td>Protease inhibitor cocktail</td><td>Beyotime</td><td>P1030</td><td>Proteinase inhibitor</td></tr><tr><td>Sodium citrate</td><td>Aladdin</td><td>7447-40-7</td><td>Cell hypotonic treatment</td></tr><tr><td>Transmission electron microscopy (TEM)</td><td>JEOL</td><td>JEM-2100plus</td><td>Morphology image</td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima XPN-100</td><td>Exosome centrifuge</td></tr><tr><td>ZetaView nanoparticle&amp;nbsp; tracking analyzers</td><td>Particle Metrix</td><td>PMX120</td><td>Nanoparticle tracking analysis</td></tr></tbody></table>

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.

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 monodispe...

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Author Spotlight: Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles 

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).

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, ...

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1.2K Views.  Tianjin University. 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). 

Video: Author Spotlight: Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles 

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Immunology and Infection

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

Setting a Successful Sorting for Extracellular Vesicle Isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

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

Summary

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Chapters in this video

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

In Vitro Polymerization of F-actin on Early Endosomes

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

An Improved Method to Isolate Mitochondrial Contact Sites

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Setting a Successful Sorting for Extracellular Vesicle Isolation