This work was supported by grants from the National Natural Science Foundation of China (32000974, 81930025, and 82170988) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU) and the Analytical and Testing Central Laboratory of Military Medical Innovation Center of Air Force Medical University.

All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-week-old C57Bl/6 mice (no preference for either females or males) were used. Cryopreserved human umbilical cord-derived MSCs (UCMSCs), used for the present study, were obtained from a commercial source (see Table of Materials). The use of human cells was approved by the Ethics Committee of Fourth Military Medical University.
1. Culture of human mesenchymal stem cells (hMSCs)
<ol>
	<li>Prepare the accessories and solutions required for the experimental work, including pipettes, pipette tips, 10 cm Petri dish, thermostatic equipment (water bath), rubber gloves, 75% alcohol, culture medium (incubated at 37 &#176;C), fatal bovine serum (FBS), and 100x penicillin-streptomycin (P/S) solution (see Table of Materials).</li>
	<li>Prepare commercially available alpha-Minimum Essential Medium (&#945;-MEM) by supplementing with 20% FBS and 1% P/S. 
	NOTE: Dilute all the solutions used for isolation and analysis of EVs in ultrapure filtered water made by the ultrapure filtered water purification system (see Table of Materials).</li>
	<li>Recover the cryopreserved human umbilical cord-derived MSCs (UCMSCs) from the liquid nitrogen by melting them in a 37 &#176;C water bath.</li>
	<li>Quickly and gently transfer the melted cell suspension into a 15 mL tube with 5 mL of culture medium (step 1.2). Centrifuge at 4 &#176;C, 500 x g for 5 min.</li>
	<li>Remove the supernatant with a sterile pipette and retain the cell precipitate. Add 2 mL of culture medium into the centrifuge tube and gently resuspend the cells.</li>
	<li>Seed the cells in a 10 cm Petri dish, and add 8 mL of culture medium to a total of 10 mL.</li>
	<li>Culture MSCs at 37 &#176;C in 5% CO2 in the cell incubator.</li>
</ol>
2. Differential centrifugation for isolation of Exos and MVs
<ol>
	<li>When MSCs grow to &#62;90% confluence, replace the culture medium with &#945;-MEM supplemented with 20% EV-depleted FBS and 1% P/S for 8 mL of each dish. 
	NOTE: Centrifuge FBS at 4 &#176;C, 1,50,000 x g overnight to remove EVs and other impurities.</li>
	<li>After 48 h of culture, collect the culture medium (step 2.1) into 50 mL centrifuge tubes.</li>
	<li>Centrifuge the MSC culture medium at 4 &#176;C, 800 x g for 10 min.</li>
	<li>Remove the cell fragments and cellular debris by transferring the supernatant to clean 1.5 mL conical tubes. 
	NOTE: Using 1.5 mL conical tubes is necessary to increase EV yield.</li>
	<li>Centrifuge the supernatant at 4 &#176;C, 16,000 x g for 30 min. The pellet is MVs. MVs from each dish of cells were resuspended with 50 &#181;L of PBS.</li>
	<li>Transfer the supernatant obtained by centrifugation in step 2.5 by pipette to clean ultracentrifuge tubes. Centrifuge at 1,50,000 x g for 2 h at 4 &#176;C.</li>
	<li>Discard the supernatant and collect the residue, which is Exos. Resuspend Exos from seven dishes of cells in 50 &#181;L of PBS. 
	NOTE: The sixth-passaged UCMSCs are used for EV isolation. To get enough MVs and Exos for experimental use, 7-8 dishes of cultured cells are usually needed. The EV samples are used immediately for the following characterization steps or for therapeutic application, which is better not stored. 
	CAUTION: Avoid repeated freezing and thawing of EVs, and it is best to store EVs at 4 &#176;C in the short term and not at -80 &#176;C.</li>
</ol>
3. Detection of particle numbers and size distribution of EVs from MSCs
NOTE: For size distribution evaluation, nanoparticle tracking analysis (NTA) is performed by a commercially available nanoparticle tracking analyzer (see Table of Materials).
<ol>
	<li>Dilute 1 &#181;L of EVs (from step 2.5 and step 2.7) in 1,499 &#181;L of PBS and mix enough in a 15 mL tube.</li>
	<li>Operate the tracking analyzer following the manufacturer&#39;s instructions. Firstly, start the compatible software (see Table of Materials).</li>
	<li>Fill the sample cell with distilled water, and then allow the instrument to start the cell check.</li>
	<li>Calibrate the instrument with a prepared standard solution. Ensure that the particle number displayed on the software detection interface is between 50-400 (preferably around 200). Click OK. 
	NOTE: Prepare the standard solution by reconstituting 1 &#181;L of calibration solution (see Table of Materials) in 1 mL of distilled water to generate a primary solution, and then take 100 &#181;L of the primary solution and add 25 mL of distilled water to prepare a standard solution (1:250,000). Store this working reagent at 4 &#176;C for a week.</li>
	<li>Rinse the sample cell with 5 mL of distilled water.</li>
	<li>Before sample analysis, flush the channel with 1 mL of distilled water. Ensure that the particle number displayed on the software detection interface is less than 10.</li>
	<li>Inject 1 mL of the EV sample prepared in step 3.1. Perform vesicle test according to the operating instructions of the instrument. 
	CAUTION: The sample and distilled water are injected with a 1 mL syringe at a constant speed of 0.5 mL/s under careful manual control.</li>
</ol>
4. Characterization of EV morphology by transmission electron microscope (TEM)
<ol>
	<li>Dilute 1 &#181;L of EVs from step 2.7 in 199 &#181;L of PBS and mix enough. Add 20 &#181;L of EV suspension drops to the formvar/carbon-coated square mesh (see Table of Materials), and let it stay for 3 min.</li>
	<li>Remove excess liquid by a small filter paper, and let it stay for 15 s to slightly dry the surface.</li>
	<li>Negatively stain EV samples with 1.5% phosphotungstic acid droplets for 40 s.</li>
	<li>Remove the excess phosphotungstic acid and allow it to stand for 15 s to slightly dry the surface.</li>
	<li>Put the sample containing formvar/carbon-coated square mesh into a clean dish covered with filter papers. Observe and capture images with a transmission electron microscope. 
	NOTE: In this process, Exos are taken as an example.</li>
</ol>
5. Labeling of MSCs-derived EVs
<ol>
	<li>After EVs extraction (step 2.5), resuspend with 250 &#181;L of PBS in a 1.5 mL conical tube.</li>
	<li>Use another 1.5 mL conical tube for preparing the working solution of the PKH26 dye; use 1 &#181;L of PKH26 labeling reagent diluted with 250 &#181;L of Diluent C (a component of the commercially available EV labeling kit used for the present study, see Table of Materials). 
	NOTE: Prepare the working solution immediately before use. 
	CAUTION: Excessive PKH26 dye or labeling without pre-dilution is at risk of damaging the EVs.</li>
	<li>Mix the resuspended EVs with the working solution. Allow it to stand at room temperature for 5 min, and then add 500 &#181;L of EV-depleted FBS to stop the reaction.</li>
	<li>For MVs, centrifuge at 4 &#176;C, 16,000 x g for 30 min, and discard the supernatant by a pipette. Add 1 mL of PBS to rinse the residue.</li>
	<li>Centrifuge at 4 &#176;C, 16,000 x g for 30 min, and discard the supernatant to remove the unbound dye.</li>
	<li>Resuspend the residue with 200 &#181;L of PBS. Drop 20 &#181;L of suspension on the slide and observe it under a fluorescence microscope. 
	NOTE: In this process, MVs are taken as an example.</li>
</ol>
6. Local transplantation and systemic injection of MSC-EVs
NOTE: For the following procedures, place the EVs on ice before injection.
<ol>
	<li>Perform local transplantation following the steps below.
	<ol>
		<li>Anesthetize the mice with 4% (vol/vol) isoflurane.&#160;Maintain anesthesia at 1%-3% isoflurane during the procedure.</li>
		<li>Before the surgery, administer 5 mg/kg carprofen (IP) for analgesia to minimize postprocedural pain.</li>
		<li>Before wound excision, shave the dorsal hair and sterilize the dorsal surface by applying 3 alternating rounds of 10% povidone-iodine and 75% ethanol.&#160;</li>
		<li>Using a biopsy punch (see Table of Materials), create a full-thickness wound of 1 cm in diameter on the dorsal skin.</li>
		<li>Resuspend the MSC-derived EVs from two dishes in 100 &#181;L of PBS and administer the injection in the subcutaneous layer of the wound bed, around each wound with four sites. 
		NOTE: An average of 100 &#181;g of EVs per mouse (EVs from two 10 cm dishes of cells) is injected. Ten 10 cm dishes of MSCs are needed to yield enough EVs to set up an experiment of n = 5 in a murine model.</li>
		<li>After treatment, wrap the mice with sterile gauze and place them on heat insulation pads (see Table of Materials) until they wake up.</li>
		<li>Ensure that the mice are not left unattended until they have regained sufficient consciousness. Do not return the mice to the company of other animals until they have fully recovered.</li>
		<li>Administer additional doses of analgesia on day 2 and day 3 post-surgery as required.&#160;</li>
	</ol>
	</li>
	<li>Perform systemic injection.
	<ol>
		<li>Resuspend MSC-derived EVs in 200 &#181;L of PBS, and then mix with heparin solution at a 10:1 volume ratio.</li>
		<li>Place the mice into the caudal vein imaging system (see Table of Materials). Press the switch to lift the lever.</li>
		<li>Disinfect the tail vein using an alcohol pad before the IV injection. Then, inject the mice systematically with suspension prepared in step 6.2.1 through the tail vein. The procedure is helped by a tail vein illustrator. 
		NOTE: Inject EV suspension immediately after mixing with heparin. An average of 100 &#181;g EVs per mouse (EVs from two 10 cm dishes of cells) is injected. Ten 10 cm dishes of MSCs are needed to yield enough EVs to set up an experiment of n = 5 in a murine model. 
		CAUTION: Mouse tail vein injection is usually limited to 200 &#181;l of volume. The injection should go slowly and stop if you see a bump or resistance, which suggests the needle is out of the vein. If the mice show&#160;adverse clinical signs like curling and trembling, this could be a shock response to high-volume injection, fast injection, or toxicity/anaphylaxis.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

EVs are emerging to play an important role in diverse biological activities, including antigen presentation, genetic material transport, cell microenvironment modification, and others. Furthermore, their wide application brings new approaches and opportunities for diagnosing and treating diseases21. Implementation of therapeutic applications of EVs is based on successful isolation and characterization. However, due to the lack of standardized isolation and purification methods and the low extracti...

Stem cells are undifferentiated pluripotent cells with self-renewal capability and translational potential1. Mesenchymal stem cells (MSCs) are easily isolated, cultured, expanded, and purified in the laboratory, which remains characteristic of stem cells after multiple passages. In recent years, increasing evidence has supported the view that MSCs act in a paracrine mode in therapeutic use2,3. Especially the secretion of extracellular vesicles (EVs) plays a crucial role in mediating the biological functions of MSCs. As heterogeneous membranous nanoparticles released from most cell types, EVs consist of subcategories termed exosomes (Exos), microvesicles (MVs), and even larger apoptotic bodies4,5. Among them, Exos is the most widely studied EV with a size of 40-150 nm, which is of an endosomal origin and actively secreted in physiological conditions. MVs are formed by shedding directly from the surface of the cell plasma membrane with a diameter of 100-1,000 nm, which are characterized by high expression of phosphatidylserine and expression of surface markers of donor cells6. EVs contain RNA, proteins, and other bioactive molecules, which have similar functions to the parent cells and play a significant role in cell communication, immune response, and tissue damage repair7. MSC-EVs have been widely investigated as a powerful cell-free therapeutic tool in regenerative medicine8.
Isolation and purification of MSC-derived EVs is a common issue in the field of research and application. At present, differential and density gradient ultracentrifugation9, ultrafiltration process10, immunomagnetic separation11, molecular exclusion chromatograph12, and microfluidic chip13 are widely employed methods in the isolation and purification of EVs. With the advantages and disadvantages of each approach, the quantity, purity, and activity of collected EVs cannot be satisfied at the same time14,15. In the present study, the differential centrifugation protocol of isolation and characterization of EVs from cultured MSCs is shown in detail, which has supported efficient therapeutic use16,17,18,19,20. The adaptability of this method for a series of downstream approaches, such as fluorescent labeling, local transplantation, and systemic injection, has further been exampled. Implementing this procedure will address the need for simple and reliable collection and application of MSC-EVs in translational research.

<table><tbody><tr><td>10% povidone-iodine (Betadine)</td><td>Weizhenyuan</td><td>10053956954292</td><td>Wound disinfection</td></tr><tr><td>Calibration solution</td><td>Particle Metrix</td><td>110-0020</td><td>Calibrate the NTA instrument</td></tr><tr><td>Carprofen</td><td>Sigma</td><td>53716-49-7</td><td>Analgesic medicine</td></tr><tr><td>Caudal vein imager&amp;nbsp;</td><td>KEW Life Science</td><td>KW-XXY</td><td>Caudal vein imager</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5418R</td><td>Centrifugation</td></tr><tr><td>Fatal bovine serum</td><td>Corning</td><td>35-081-CV</td><td>Culture of UCMSCs</td></tr><tr><td>Formvar/carbon-coated square mesh</td><td>PBL Assay Science&amp;nbsp;</td><td>24916-25</td><td>Transmission electron microscope</td></tr><tr><td>Heating pad</td><td>Zhongke Life Science</td><td>Z8G5JBMz</td><td>Post-treatment care of animals</td></tr><tr><td>Heparin Solution</td><td>StemCell</td><td>7980</td><td>Systemic injection</td></tr><tr><td>Isoflurane</td><td>RWD Life Science</td><td>R510-22</td><td>Animal anesthesia</td></tr><tr><td>Minimum Essential Medium Alpha basic (1x)</td><td>Gibco</td><td>C12571500BT</td><td>Culture of UCMSCs</td></tr><tr><td>Nanoparticle tracking analyzer</td><td>Particle Metrix</td><td>ZetaView PMX120</td><td>Nanoparticle tracking analysis</td></tr><tr><td>PBS (1x)</td><td>Meilunbio</td><td>MA0015</td><td>Resuspend EVs</td></tr><tr><td>Penicillin/Streptomycin</td><td>Procell Life Science</td><td>PB180120</td><td>Culture of UCMSCs</td></tr><tr><td>Phosphotungstic acid</td><td>Solarbio</td><td>12501-23-4</td><td>Transmission electron microscope</td></tr><tr><td>Pipette</td><td>Eppendorf</td><td>3120000224</td><td></td></tr><tr><td>PKH26 Red Fluorescent Cell Linker Kit</td><td>Sigma-Aldrich</td><td>MINI26</td><td>Labeling EVs</td></tr><tr><td>Skin biopsy punch</td><td>Acuderm</td><td>69038-10-50</td><td>Skin defects</td></tr><tr><td>Software ZetaView</td><td>Particle Metrix</td><td>Version 8.05.14 SP7&amp;nbsp;</td><td></td></tr><tr><td>Thermostatic equipment</td><td>Grant</td><td>v-0001-0005</td><td>Water bath</td></tr><tr><td>Transmission electron microscope</td><td>HITACHI</td><td>HT7800</td><td>Transmission electron microscope</td></tr><tr><td>UCMSCs</td><td>Bai&#039;ao&amp;nbsp;</td><td>UKK220201</td><td>Commercially UCMSCs</td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>XPN-100</td><td>Centrifugation</td></tr><tr><td>Ultrapure filtered water purification system</td><td>Milli-Q</td><td>IQ 7000</td><td>Preparation of ultrapure water</td></tr></tbody></table>

isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

MVs and Exos from cultured human UCMSCs are isolated following the experimental workflow (Figure 1). The NTA results demonstrate that the size of Exos from human MSCs ranges from 40 nm to 335 nm with a peak size of about 100 nm, and the size of MVs ranges from 50 nm to 445 nm with a peak size of 150 nm (Figure 2). Morphological characterization of MSC-derived Exos exhibit a typical cup shape (Figure 3). EVs are efficiently labeled b...

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Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

isolation-characterization-therapeutic-application-extracellular

Research

JoVE Journal

Biology

1.9K Views.  Northwest University. Deze procedure voorzag in de behoefte aan eenvoudige en betrouwbare mesenchymale stamcel-afgeleide extracellulaire blaasjesverzameling, en van toepassing bij de vertaling van onderzoek. Het verzamelen van extracellulaire blaasjes door differentiële centrifugatie is eenvoudig, gemakkelijk schaalbaar en reproduceerbaar, wat nuttig zal zijn voor het veld. Extracellulaire blaasjes onduidelijk}van onduidelijk}mesenchymale stamcel is een onduidelijke}toepassing in moderne ziektebehandelingen.

Video: Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

Bioengineering

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Medicine

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

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. ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

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