This work was partially supported by the National Natural Science Foundation of China (82202473).

The overview of the protocol steps is shown in Figure 1. The details of the reagents and equipment used in the study are listed in the Table of Materials.
1. Media preparation
<ol>
	<li>Prepare a culture medium for ADSC without exosomes using 450 mL of basal culture medium, 50 mL of fetal bovine serum without exosomes, and 5 mL of triple antibiotics.</li>
</ol>
2. Resuscitating and culturing ADSCs
NOTE: Resuscitate ADSCs.
<ol>
	<li>Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.</li>
	<li>Preheat the medium in a 37 &#176;C water bath beforehand.</li>
	<li>Retrieve 2 tubes of frozen ADSCs (P3) from liquid nitrogen and agitate them in a 37 &#176;C water bath with constant temperature until fully thawed. 
	NOTE: The opening of the freezer tube should not be in contact with water to prevent contamination.</li>
	<li>Disinfect the cryopreservation tube using 75% alcohol and then move it to the ultra-clean table. Aspirate the cell suspension using a pipette and place it in a 15 mL centrifuge tube.</li>
	<li>Place the centrifuge tube into a low-speed centrifuge machine and centrifuge at 250 x g for 5 min at 4 &#176;C.</li>
	<li>Prepare four 10 cm culture dishes and put 9 mL of medium into each dish. Mark the name, cell type, generation, and experimental date, and then preheat them in a 37 &#176;C thermostatic cell incubator.</li>
	<li>Disinfect the centrifuge tube and move it to the ultra-clean table. Remove the supernatant with a pipette.</li>
	<li>Add 4 mL of medium and gently agitate the solution (1 x 105 cells/mL) until it is evenly dispersed. 
	NOTE: Take care not to eliminate the cell pellet when extracting the supernatant.</li>
	<li>Add 1 mL of cell suspension to each dish. Shake the dishes with a cross method. 
	NOTE: Position the plate on a level surface and agitate it in a cross pattern (moving it up, down, left, and right) for 5-6 repetitions. Avoid circular motions, as they may cause cell accumulation in the center.</li>
	<li>Observe the cells at 40x magnification with the optical microscope, and then place the dishes into the incubator. 
	NOTE: The primary purpose of observing the cells is to determine if they are evenly distributed, as uneven distribution will affect subsequent growth.</li>
	<li>Prepare exchange medium for the cells.
	<ol>
		<li>Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.</li>
		<li>Preheat the medium in a 37 &#176;C water bath beforehand.</li>
		<li>Observe the cell state under the optical microscope at 40x magnification. The confluence level is approximately 50%. 
		NOTE: The confluence level is calculated as the ratio of the area occupied by the cells to the surface area of the petri dish after the cells adhere to the wall and fully stretch.</li>
		<li>Transfer the culture dish to the ultra-clean table (Biosafety level 2) after disinfection and remove the medium.</li>
		<li>Rinse each culture dish twice with 2 mL of PBS solution gently.</li>
		<li>Add 10 mL of medium to each dish. Transfer the dishes to the incubator.</li>
	</ol>
	</li>
</ol>
3. Collecting the ADSCs supernatant and extracting the EVs
<ol>
	<li>Turn on the UV radiation of the ultra-clean table beforehand and disinfect it for 30 min.</li>
	<li>Observe the cell state under a microscope. The confluence level is approximately 80%.</li>
	<li>Transfer the dish to the ultra-clean table after disinfection. Carefully collect the supernatant with a pipette and place it in a 50 mL centrifuge tube. 
	NOTE: Cells can be either frozen or discarded. If sterility is not required, the following steps can be performed on the operating table. At this point, the experiment can be paused and then restarted later. If the experiment is halted, store the supernatant in a 4 &#176;C refrigerator for not more than one week. Long-term storage is not recommended as it may affect the activity of EVs. The optimal approach is to extract EVs immediately.</li>
	<li>Centrifuge (300 x g, 10 min, at room temperature) to remove suspended cells. Collect the supernatant with a pipette. 
	NOTE: Do not pour. Leave a little supernatant to prevent the pellet from being sucked up.</li>
	<li>Centrifuge to remove cellular debris (2000 x g, 10 min, 4 &#176;C), then continue to collect the supernatant with a pipette. 
	NOTE: The key points are the same as in step 3.4.</li>
	<li>Filter the supernatant with a 0.22 um membrane to remove large particles and cell debris. 
	NOTE: Filter the supernatant with 2 mL of sterile PBS to moisten the filter membrane prior to filtration.</li>
	<li>Centrifuge in the ultracentrifuge machine (10,000 x g, 30 min, 4 &#176;C).</li>
	<li>Next, centrifuge at 1,00,000 x g for 70 min at 4 &#176;C to remove the pellet and collect the supernatant. 
	NOTE: The obtained EVs were purified by centrifugation multiple times to increase their purity. The pellet may not be visible to the naked eye. Therefore, it is crucial to operate the pipette gently and to leave a little supernatant at the bottom to avoid pellet collection.</li>
	<li>Remove the supernatant with a pipette. Resuspend the pellet with PBS and centrifuge (1,00,000 x g, 70 min, 4 &#176;C). 
	NOTE: The pellet may not be visible to the naked eye. Therefore, when aspirating the supernatant with a pipette, it is critical to gently and thoroughly remove the supernatant to maximize the purity of EVs.</li>
	<li>Remove the supernatant and obtain the EVs pellet. Resuspend it with 1 mL of PBS, transfer it into a 1 mL centrifuge tube, and store it in a 4 &#176;C refrigerator for subsequent detection. 
	NOTE: The pellet may not be visible to the naked eye. Consequently, when aspirating the supernatant with a pipette, it is crucial to gently and thoroughly remove the supernatant to maximize EVs purity. If the samples are not needed for an extended period, store them in a -80 &#176;C refrigerator.</li>
</ol>

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+from+adipose+tissue-a+potential+role+in+obesity+and+type+2+diabetes.">Extracellular vesicles from adipose tissue-a potential role in obesity and type 2 diabetes.</a> Front Endocrinol (Lausanne). 8, 202 (2017).</li><li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+biological+effects+of+extracellular+vesicles+from+adipose-derived+stem+cells+were+markedly+increased+by+low-intensity+ultrasound+stimulation+for+promoting+diabetic+wound+healing).">Production and biological effects of extracellular vesicles from adipose-derived stem cells were markedly increased by low-intensity ultrasound stimulation for promoting diabetic wound healing).</a> Stem Cell Rev Rep. 19 (3), 784-806 (2023).</li><li>Mou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+from+human+adipose-derived+stem+cells+for+the+improvement+of+angiogenesis+and+fat-grafting+application.">Extracellular vesicles from human adipose-derived stem cells for the improvement of angiogenesis and fat-grafting application.</a> Plast Reconstr Surg. 144 (4), 869-880 (2019).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+bone+immobilized+with+human+adipose+stem+cells-derived+exosomes+promotes+bone+regeneration.">Tissue-engineered bone immobilized with human adipose stem cells-derived exosomes promotes bone regeneration.</a> ACS Appl Mater Interfaces. 10 (6), 5240-5254 (2018).</li><li>Han, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell-derived+extracellular+vesicles+inhibit+the+fibrosis+of+fibrotic+buccal+mucosal+fibroblasts+via+the+microRNA-375/foxf1+axis.">Adipose-derived stem cell-derived extracellular vesicles inhibit the fibrosis of fibrotic buccal mucosal fibroblasts via the microRNA-375/foxf1 axis.</a> Stem Cells Int. 2021, 9964159 (2021).</li><li>Brezgin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+aspects+of+manufacturing+and+analytical+control+of+biological+nanoparticles.">Technological aspects of manufacturing and analytical control of biological nanoparticles.</a> Biotechnol Adv. 64, 108122 (2023).</li><li>Kim, S. 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The authors have no financial interest to declare.

During the formal experimental process, several points are crucial for achieving the best experimental results. Based on our previous experience, it is recommended to opt for passage 3-6 ADSCs, as they ensure the best possible cell state. Before P3, red blood cells, endothelial cells, and other miscellaneous cells may not have been screened out. After P6, the cells may gradually age, which can affect the state of secreted EVs. Secondly, the supernatant must be collected when the cell confluence degree is between 80% and ...

Most ADSCs are derived from adipose tissue and have been demonstrated to promote the regeneration and reconstruction of various tissues and organs, including the myocardium, bone, and skin1. Recent research suggests that the regenerative function of ADSCs may be due to intercellular contact and the paracrine effects of the cells2. The paracrine effect is an important means for cells to interact and transfer information over short distances, and this function is achieved through extracellular vesicles (EVs).
EVs are double-layer membrane structures produced by cells, with a diameter ranging from 40 nm to 160 nm (with an average of about 100 nm). They affect different cellular functions, such as cytokine production, cell proliferation, apoptosis, and metabolism3,4. Numerous studies have been conducted on the functions of ADSC EVs, including promoting bone regeneration, oral mucosal tissue regeneration, adipose tissue survival after tissue transplantation, and skin wound repair5,6,7,8. The enormous potential of ADSC EVs in regenerative medicine is evident. Various methods exist for extracting and separating EVs from the supernatant, such as techniques based on centrifugation, precipitation, molecular size, affinity, and microfluidics. The ultracentrifugation method is widely considered the gold standard for isolating EVs9. The fundamental principle of ultracentrifugation for EV separation is based on the fact that particles in the sample have varying sedimentation coefficients, resulting in their sedimentation and aggregation in distinct separation layers. Nevertheless, a detailed protocol emphasizing precautions during ultracentrifugation has not yet been established.
Therefore, this study objectively outlines the ADSC culture, supernatant collection, and EVs ultracentrifugation procedures and key points with a logical flow of information and clear, formal language. This provides a valuable reference for future experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrodisc Needle Filter of Supor Membrane</td>
			<td>Acros Organics</td>
			<td>4652</td>
			<td>0.22 &#956;m</td>
		</tr>
		<tr>
			<td>Basal Medium For Cell Culture</td>
			<td>OriCell</td>
			<td>BHDM-03011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Without EXO + Culture Supplement (For Human Adipose-derived Mesenchymal Stem Cells)</td>
			<td>OriCell</td>
			<td>HUXMD-05002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>Lx70-S8F2</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Speed Centrifuge</td>
			<td>Anhui USTC Zonkia Scientific Instruments Co.,Ltd.</td>
			<td>SC-3612</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima Max-XP Tabletop Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Gentamicin Solution (100x)</td>
			<td>Solarbio</td>
			<td>P1410</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Our research focuses on the preparation and purification of extracellular vesicles from human adipose-derived mesenchymal stem cells. The problem we want to solve is how to standardize this process and improve the purification efficiency. Currently, there are many technologies used to extract extracellular vesicles, such as ultracentrifugation, ultrafiltration, volume exclusion chromatography, polymer precipitation, immunoaffinity capture, microfluidic technology, and so on.However, ultracentrifugation is recognized as a gold standard, admittedly. In the future, we will focus on the application of extracellular vesicles as vectors, including carrying MRNA, plasmids, and other small molecules and transfecting them into cells.

Firstly, ADSCs were characterized and identified, including their morphology10 and surface antibodies. Based on Figure 2A, it is apparent that ADSCs are arranged in a spindle shape and form a vortex after dense growth. The cultured cells were differentiated into adipogenic, osteogenic, and chondrogenic cells and stained with Oil Red O, Alizarin Red, and Alcian Blue11. The induced differentiation experiment and flow cytometry validated that they...

Read this Scientific Article Protocol about Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs at JoVE.com

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 ...

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392 Views.  Chinese PLA General Hospital. Our research focuses on the preparation and purification of extracellular vesicles from human adipose-derived mesenchymal stem cells. The problem we want to solve is how to standardize this process and improve the purification efficiency. Currently, there are many technologies used to extract extracellular vesicles, such as ultracentrifugation, ultrafiltration, volume exclusion chromatography, polymer precipitation, immunoaffinity capture, microfluidic technology, and so on.However, ul...

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

Resuscitation and Culture of Adipose-Derived Mesenchymal Stem Cells (ADSCs) for Extracellular Vesicle Extraction

To begin, retrieve two cryopreservation tubes containing frozen human adipose-derived mesenchymal stem cells at passage three from liquid nitrogen. Agitate them in a 37 degree Celsius water bath until fully thawed. Then disinfect the tubes using 75%alcohol.Place the disinfected tubes on an ultra-clean table. Using a pipette, aspirate the cell suspension and transfer it to a 15 milliliter centrifuge tube. Centrifuge the tube in a low speed centrifuge at 250 G and four degrees Celsius for five minutes.Meanwhile, prepare four 10-centimeter culture dishes and add nine milliliters of medium to each dish. Mark the name, cell type, generation, and the experiment date on each dish. Then preheat them in a 37 degree Celsius thermostatic cell incubator.Once the five minute centrifugation is over, disinfect the centrifuge tube before moving it to an ultra-clean table. Using a pipette, remove the supernatant and add four milliliters of medium to achieve the desired cell concentration. Gently agitate the solution until it is evenly dispersed.Next, add one milliliter of cell suspension to each preheated dish and shake it by agitating the same on a level surface in a cross pattern. Observe the cells at 40x magnification under an optical microscope before placing the dishes into the 37 degree Celsius incubator. On day two, observe the cell state under the optical microscope at 40x magnification before proceeding for exchange medium preparation.After disinfection, transfer the culture dishes to an ultra-clean table with Biosafety Level 2. Remove the medium from each culture dish. Rinse the dish gently with two milliliters of PBS solution twice and add 10 milliliters of medium to each dish.Transfer the cells to the incubator until they're ready for supernatant collection.

Extracting Extracellular Vesicles (EVs) from Adipose-Derived Mesenchymal Stem Cell Culture

To begin, collect the supernatant from the human adipose-derived mesenchymal stem cell culture once the cells are 80%confluent. Collect the supernatant carefully using a pipette and transfer it to a 50 milliliter centrifuge tube. Centrifuge the tube at 300 x g for 10 minutes at room temperature to remove suspended cells, then use a pipette to collect the supernatant without disturbing the pellet.Centrifuge the supernatant at 2000 x g for 10 minutes at four degrees Celsius to remove cellular debris. Collect the resultant supernatant with a pipette and filter it through a 0.22 micron membrane to remove large particles and cell debris. Subject the filtrate to ultra centrifugation at 10, 000 x g for 30 minutes at four degrees Celsius.Then perform a final centrifugation at 100, 000 x g for 70 minutes at four degrees Celsius. Using a pipette, remove the supernatant without collecting the pellet. Resuspend the pellet in PBS before centrifuging the tube at 100, 000 x g for 70 minutes at four degrees Celsius.Remove the supernatant and resuspend the obtained pellet of extracellular vesicles, or EVs, in one milliliter of PBS. Finally, transfer the EV suspension to a one milliliter centrifuge tube and store it at four degrees Celsius until further analysis. Transmission electron microscopy clearly revealed the cup-shaped structure of the EVs with a bilayer membrane.Nanoparticle tracking analysis indicated that they have a particle size distribution of around 50 to 150 nanometers. Western blot analysis showed that the characteristic marker proteins of EVs were expressed smoothly.