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

<ol>
	<li>Al-Ghadban, S., Artiles, M., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+stem+cells+in+regenerative+medicine:+Looking+forward.">Adipose stem cells in regenerative medicine: Looking forward.</a> Front Bioeng Biotechnol. 9, 837464 (2021).</li><li>Xin, H., Li, Y., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes/miRNAs+as+mediating+cell-based+therapy+of+stroke.">Exosomes/miRNAs as mediating cell-based therapy of stroke.</a> Front Cell Neurosci. 8, 377 (2014).</li><li>Li, Q., 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=The+tissue+origin+effect+of+extracellular+vesicles+on+cartilage+and+bone+regeneration.">The tissue origin effect of extracellular vesicles on cartilage and bone regeneration.</a> Acta Biomater. 125, 253-266 (2021).</li><li>Gao, X., Salomon, C., Freeman, D. 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. H., 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=Character+comparison+of+abdomen-derived+and+eyelid-derived+mesenchymal+stem+cells.">Character comparison of abdomen-derived and eyelid-derived mesenchymal stem cells.</a> Cell Prolif. 46 (3), 291-299 (2013).</li><li>Gan, L., 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=Exosomes+from+adipose-derived+mesenchymal+stem+cells+improve+liver+fibrosis+by+regulating+the+miR-20a-5p/TGFBR2+axis+to+affect+the+p38+MAPK/NF-&#954;B+pathway.">Exosomes from adipose-derived mesenchymal stem cells improve liver fibrosis by regulating the miR-20a-5p/TGFBR2 axis to affect the p38 MAPK/NF-&#954;B pathway.</a> Cytokine. 172, 156386 (2023).</li><li>Jung, M. K., Mun, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+and+imaging+of+exosomes+by+transmission+electron+microscopy.">Sample preparation and imaging of exosomes by transmission electron microscopy.</a> J Vis Exp. (131), e56482 (2018).</li><li>Han, Y. D., 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=Co-transplantation+of+exosomes+derived+from+hypoxia-preconditioned+adipose+mesenchymal+stem+cells+promotes+neovascularization+and+graft+survival+in+fat+grafting.">Co-transplantation of exosomes derived from hypoxia-preconditioned adipose mesenchymal stem cells promotes neovascularization and graft survival in fat grafting.</a> Biochem Biophys Res Commun. 497 (1), 305-312 (2018).</li><li>Linares, R., Tan, S., Gounou, C., Arraud, N., Brisson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+centrifugation+induces+aggregation+of+extracellular+vesicles.">High-speed centrifugation induces aggregation of extracellular vesicles.</a> J Extracell Vesicles. 4, 29509 (2015).</li><li>Lamparski, H. 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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 90% to ensure sufficient secretion of as many EVs as possible. To maintain the maximum purity of EVs, the researchers must be cautious during the first three centrifugations and ensure no liquid residue is left to prevent impurities and pellets from aspirating. During the last two centrifugations, ensure that the supernatant is completely removed while protecting the pellet.
Ultracentrifugation is cost-effective and requires no further treatment. This technique has been widely used in EV studies and has demonstrated satisfactory efficiency in obtaining EVs, as confirmed by characterization experiments. Nonetheless, there are some inevitable drawbacks. First, the EVs recovery rate is low. Second, EVs tend to aggregate sometimes. Additionally, non-cellular components can be isolated, and some EVs may rupture during the process, leading to debris accumulation14,15,16. To address these defects, most research focuses on purifying EVs12,13. However, this protocol covers the entire process from cell culture to exosome purification because we believe that the entire process impacts the status and purity of the final extracted EVs. It is crucial to adhere to the aforementioned guidelines during operation. Additionally, collecting more supernatant and implementing 3D cell culture may be beneficial, as numerous studies have demonstrated that 3D culture is more effective in stimulating the secretion of EVs compared to 2D culture17,18.
This protocol outlines the process of extracting EVs from ADSCs using ultracentrifugation. It provides practical guidance and enables experimenters to avoid details during the operation as much as possible. In summary, the protocol assists experimenters in improving the quality of samples and serves as a reminder that we should not only focus on the details of each step but also consider the experiment as a whole.

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.

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 are indeed ADSCs with the potential for multi-directional differentiation. However, 22% of cells are CD45 positive in the results, which won&#39;t affect the conclusion as sufficient positive and negative antibodies support it (Figure 2A,B).
Next, the identification of the extracted EVs is essential. It mainly includes three aspects: morphology, particle size, and marker proteins. The TEM results clearly reveal the cup-shaped EV structure with a bilayer membrane12 (Figure 3A). NTA indicates that EVs have a particle size distribution of around 50-150 nm, with a concentration of approximately 1.6 x 1010 particles/mL (Figure 3B). Western Blot analysis also shows that the marker proteins of EVs, including CD9, CD63, and TSG10113, are expressed smoothly (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66585/66585fig01v2.jpg" /> 
Figure 1: Overview of the protocol steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66585/66585fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66585/66585fig02.jpg" /> 
Figure 2: Characterization of ADSCs. (A) The morphology of ADSCs is fusiform. ADSCs were cultured in the medium for adipogenic, osteogenic, and chondrogenic differentiation, respectively. Adipogenic differentiation was identified by Oil Red O staining. Osteogenic and chondrogenic differentiation was confirmed by Alizarin Red staining and Alcian Blue staining, respectively. Scale bar: 100 &#181;m. (B) Flow cytometry analysis of ADSCs for MSC surface markers (CD90, CD105, CD29, and CD73) and hematopoietic cell-specific markers (CD31, CD34, CD45, and HLA-DR). The gating strategy: circle the blank group first. Based on that, circle CD90, CD105, CD29, CD73, CD31, CD34, CD45, and HLA-DR successively according to the labeled antibodies of each group. This figure is adapted from Han, Y. D. et al.13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66585/66585fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66585/66585fig03.jpg" /> 
Figure 3: Characterization of EVs. (A) The TEM results clearly reveal the cup-shaped EV structure of the bilayer membrane. Scale bar: 200 nm. (B) NTA indicates that EVs have a particle size distribution of around 50-150 nm, with a concentration of approximately 1.6 x 1010 particles/ mL. (C) Detection of TSG101, CD9, and CD63 expressions in exosomes by Western blot. This figure is adapted from Han, Y. D. et al.13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66585/66585fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

purification-characterization-extracellular-vesicles-from-human

Nanyang Technological University

Research

JoVE Journal

Medicine

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

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

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...