National Natural Science Foundation of China (No. 81972116, No. 81972085, No. 81772394); Key Program of Natural Science Foundation of Guangdong Province (No.2018B0303110003); Guangdong International Cooperation Project (No.2021A0505030011); Shenzhen Science and Technology Projects (No. GJHZ20200731095606019, No. JCYJ20170817172023838, No. JCYJ20170306092215436, No. JCYJ20170413161649437); China Postdoctoral Science Foundation (No.2020M682907); Guangdong Basic and Applied Basic Research Foundation (No.2021A1515010985); Sanming Project of Medicine in Shenzhen (SZSM201612079); Special Funds for the Construction of High-level Hospitals in Guangdong Province.

This study was approved by the Human Ethics Committee of Shenzhen Second People&#39;s Hospital. A schematic diagram of exosomes isolated from hSF-MSCs in vitro protocol is shown in Figure 1.
1. Human SF-MSCs culture and identification
<ol>
	<li>Harvest 20 mL of SF using a syringe and needle from clinical OA patients.
	<ol>
		<li>Disinfect the knee joint of the OA patient. Puncture from the quadriceps femoris tendon outside the patella i...

<ol>
	<li>Cross, M., 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+global+burden+of+hip+and+knee+osteoarthritis:+estimates+from+the+global+burden+of+disease+2010+study.">The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study.</a> Annals of the Rheumatic Diseases. 73 (7), 1323-1330 (2014).</li><li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis &amp; Rheumatology. 64 (6), 1697-1707 (2012).</li><li>Huey, D. J., Hu, J. C., Athanasiou, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlike+bone,+cartilage+regeneration+remains+elusive.">Unlike bone, cartilage regeneration remains elusive.</a> Science. 338 (6109), 917-921 (2012).</li><li>Lu, J., 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=Increased+recruitment+of+endogenous+stem+cells+and+chondrogenic+differentiation+by+a+composite+scaffold+containing+bone+marrow+homing+peptide+for+cartilage+regeneration.">Increased recruitment of endogenous stem cells and chondrogenic differentiation by a composite scaffold containing bone marrow homing peptide for cartilage regeneration.</a> Theranostics. 8 (18), 5039-5058 (2018).</li><li>Ogura, T., Bryant, T., Merkely, G., Mosier, B. A., Minas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+analysis+of+revision+autologous+chondrocyte+implantation+for+failed+ACI.">Survival analysis of revision autologous chondrocyte implantation for failed ACI.</a> American Journal of Sports Medicine. 47 (13), 3212-3220 (2019).</li><li>Welch, T., Mandelbaum, B., Tom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+past,+present,+and+future.">Autologous chondrocyte implantation: past, present, and future.</a> Sports Medicine and Arthroscopy Review. 24 (2), 85-91 (2016).</li><li>McGonagle, D., Baboolal, T. G., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+joint-resident+mesenchymal+stem+cells+for+cartilage+repair+in+osteoarthritis.">Native joint-resident mesenchymal stem cells for cartilage repair in osteoarthritis.</a> Nature Reviews Rheumatology. 13 (12), 719-730 (2017).</li><li>Jo, C. 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=Intra-articular+injection+of+mesenchymal+stem+cells+for+the+treatment+of+osteoarthritis+of+the+knee:+a+proof-of-concept+clinical+trial.">Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial.</a> Stem Cells. 32 (5), 1254-1266 (2014).</li><li>Pers, Y. M., Ruiz, M., No&#235;l, D., Jorgensen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+the+management+of+inflammation+in+osteoarthritis:+state+of+the+art+and+perspectives.">Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives.</a> Osteoarthritis Cartilage. 23 (11), 2027-2035 (2015).</li><li>Neybecker, P., 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=In+vitro+and+in+vivo+potentialities+for+cartilage+repair+from+human+advanced+knee+osteoarthritis+synovial+fluid-derived+mesenchymal+stem+cells.">In vitro and in vivo potentialities for cartilage repair from human advanced knee osteoarthritis synovial fluid-derived mesenchymal stem cells.</a> Stem Cell Research &amp; Therapy. 9 (1), 329 (2018).</li><li>Jia, Z., 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=Magnetic-activated+cell+sorting+strategies+to+isolate+and+purify+synovial+fluid-derived+mesenchymal+stem+cells+from+a+rabbit+model.">Magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model.</a> Journal of Visualized Experiments: JoVE. (138), (2018).</li><li>Phinney, D. G., Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+MSC-derived+exosomes+for+cell-free+therapy.">Concise review: MSC-derived exosomes for cell-free therapy.</a> Stem Cells. 35 (4), 851-858 (2017).</li><li>Phan, J., 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=Engineering+mesenchymal+stem+cells+to+improve+their+exosome+efficacy+and+yield+for+cell-free+therapy.">Engineering mesenchymal stem cells to improve their exosome efficacy and yield for cell-free therapy.</a> Journal of Extracellular Vesicles. 7 (1), 1522236 (2018).</li><li>Dominici, M., 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+international+society+for+cellular+therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Lv, F. -. J., 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=Concise+review:+the+surface+markers+and+identity+of+human+mesenchymal+stem+cells.">Concise review: the surface markers and identity of human mesenchymal stem cells.</a> Stem Cells. 32 (6), 1408-1419 (2014).</li><li>Samsonraj, R. M., 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=Concise+review:+Multifaceted+characterization+of+human+mesenchymal+stem+cells+for+use+in+regenerative+medicine.">Concise review: Multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine.</a> Stem Cells Translational Medicine. 6 (12), 2173-2185 (2017).</li><li>Han, 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=Mesenchymal+stem+cells+for+regenerative+medicine.">Mesenchymal stem cells for regenerative medicine.</a> Cells. 8 (8), (2019).</li><li>Zhang, G., 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+derived+from+human+neural+stem+cells+stimulated+by+interferon+gamma+improve+therapeutic+ability+in+ischemic+stroke+model.">Exosomes derived from human neural stem cells stimulated by interferon gamma improve therapeutic ability in ischemic stroke model.</a> Journal of Advanced Research. 24, 435-445 (2020).</li><li>Zhou, P., 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=Migration+ability+and+Toll-like+receptor+expression+of+human+mesenchymal+stem+cells+improves+significantly+after+three-dimensional+culture.">Migration ability and Toll-like receptor expression of human mesenchymal stem cells improves significantly after three-dimensional culture.</a> Biochemical and Biophysical Research Communications. 491 (2), 323-328 (2017).</li><li>Cheng, N. C., Wang, S., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+spheroid+formation+of+human+adipose-derived+stem+cells+on+chitosan+films+on+stemness+and+differentiation+capabilities.">The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities.</a> Biomaterials. 33 (6), 1748-1758 (2012).</li><li>Guo, L., Zhou, Y., Wang, S., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+changes+of+mesenchymal+stem+cells+in+three-dimensional+(3D)+spheroids.">Epigenetic changes of mesenchymal stem cells in three-dimensional (3D) spheroids.</a> Journal of Cellular and Molecular Medicine. 18 (10), 2009-2019 (2014).</li><li>Zhang, 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=Systemic+administration+of+cell-free+exosomes+generated+by+human+bone+marrow+derived+mesenchymal+stem+cells+cultured+under+2D+and+3D+conditions+improves+functional+recovery+in+rats+after+traumatic+brain+injury.">Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury.</a> Neurochemistry International. 111, 69-81 (2017).</li><li>Cao, J., 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=Three-dimensional+culture+of+MSCs+produces+exosomes+with+improved+yield+and+enhanced+therapeutic+efficacy+for+cisplatin-induced+acute+kidney+injury.">Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury.</a> Stem Cell Research &amp; Therapy. 11 (1), 206 (2020).</li></ol>

The mesenchymal stem cells have been widely used in regenerative medicine due to their self-renewal, differentiated into tissue cells with specialized functions, and paracrine effects16,17. Notably, the paracrine effects exerted by exosomes have attracted much attention18. Exosomes carry the bio-information of MSCs and perform their biological function and overcome MSC shortcomings, such as troublesome storage and shipment. Thus, exosomes ...

Osteoarthritis (OA), resulting from joint cartilage and underlying bone breakdown, remains a severe challenge leading to disability1,2. Without blood and nerve supply, cartilage self-healing ability is minimal once being injured3,4. In the past decades, therapies based on autologous chondrocyte implantation (ACI) have made some progress in OA treatment5. For chondrocyte isolation and expansion, harvesting small cartilage from the OA joint&#39;s non-weight bearing area is necessary, causing injuries to the cartilage. Also, the procedure will require a second operation to implant the expanded chondrocytes6. Thus, one-step therapies for OA treatment without cartilage injuries are under extensive exploration.
Mesenchymal stem cells (MSCs) have been suggested as promising alternatives for OA treatment7,8. Originating from multiple tissues, MSCs can differentiate into chondrocytes with specific stimulation. Importantly, MSCs can modulate immune responses via anti-inflammation9. Therefore, MSCs hold significant advantages in OA treatment by repairing cartilage defects and modulating the immune response, especially in the inflammation milieu. For OA treatment, MSCs from synovial fluid (SF-MSCs) have recently attracted much attention due to their stronger chondrocyte differentiation ability than other MSC sources10,11. Notably, at the orthopedic clinic, the extraction of inflammatory SF from the joint cavity is a routine therapy to relieve the pain symptom of OA patients. Extracted inflammatory SF usually is disposed of as medical waste. Both patients and doctors are ready to consider autologous MSCs isolated from the inflammatory SF as OA treatment with very few ethical conflicts. However, SF-MSC therapy is compromised due to tumorigenic risks, long-time storage, and distant shipment barriers.
Exosomes, secreted by many cell types, including MSCs, carry most of the parent cell bio-information. It has been investigated in-depth as a cell-free therapy12,13. According to the updated resources available on the clinical trial government (ClinicalTrials.gov) website, more extensive exosome clinical studies are initiated and undertaken in the research fields of cancer, hypertension, and neuro-degenerative diseases. SF-MSC exosome treatment could be an exciting and challenging trial to cope with OA. Good manufacturing practice (GMP)-grade and large-scale exosome production are essential for clinical translation. Small-scale exosome isolation has been widely performed based on two-dimensional (2D) cell culture. However, large-scale exosome production strategies need optimization. A large-scale exosome manufacturing method was developed in this study, based on massive SF-MSC culture in xeno-free conditions. After ultracentrifugation from cell culture supernatants, exosome safety and function were validated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA assay kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Protein concentration assay</td>
		</tr>
		<tr>
			<td>Blood agar plate</td>
			<td>Nanjing Yiji Biochemical Technology Co. , Ltd.</td>
			<td>P0903</td>
			<td>Bacteria culture</td>
		</tr>
		<tr>
			<td>CD105 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1243C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD34 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1143C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD45 antibody</td>
			<td>BD Bioscience</td>
			<td>555483</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>Abclonal</td>
			<td>&#160;A5271</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD73 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1242C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>ABclonal</td>
			<td>&#160;A5270</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>Abclonal</td>
			<td>&#160;A1703</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD90 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1167C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo</td>
			<td></td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Confocal laser scanning fluorescence microscopy</td>
			<td>ZEISS</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytodex</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Microcarrier</td>
		</tr>
		<tr>
			<td>Dil</td>
			<td>ThermoFisher</td>
			<td>D1556</td>
			<td>Exosome label</td>
		</tr>
		<tr>
			<td>EZ-PCR Mycoplasma detection kit</td>
			<td>BI</td>
			<td>20-700-20</td>
			<td>Mycoplasma detection</td>
		</tr>
		<tr>
			<td>Flowcytometry</td>
			<td>Beckman</td>
			<td></td>
			<td>MSC identification</td>
		</tr>
		<tr>
			<td>Gene Pulser II System</td>
			<td>Bio-Rad Laboratories</td>
			<td>1652660</td>
			<td>Gene transfection</td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0.2</td>
			<td>GraphPad Software, Inc.</td>
			<td>Version 8.0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1111C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Lowenstein-Jensen culture medium</td>
			<td>Nanjing Yiji Biochemical Technology Co. , Ltd.</td>
			<td>T0573</td>
			<td>Mycobacterium tuberculosis culture</td>
		</tr>
		<tr>
			<td>MesenGro</td>
			<td>StemRD</td>
			<td>MGro-500</td>
			<td>MSC culture</td>
		</tr>
		<tr>
			<td>Nanosight NS300</td>
			<td>Malvern</td>
			<td>Nanosight NS300</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>NTA 2.3 software</td>
			<td>Malvern</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>Odyssey FC</td>
			<td>Gene Company Limited</td>
			<td></td>
			<td>Fluorescent western blotting</td>
		</tr>
		<tr>
			<td>OptiPrep electroporation buffer</td>
			<td>Sigma</td>
			<td>D3911</td>
			<td>Gene transfection</td>
		</tr>
		<tr>
			<td>Protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td>Proteinase inhibitor</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Qiagen</td>
			<td>158924</td>
			<td>Removal of RNA</td>
		</tr>
		<tr>
			<td>Sabouraud agar plate</td>
			<td>Nanjing Yiji Biochemical Technology Co., Ltd.</td>
			<td>P0919</td>
			<td>Fungi culture</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td></td>
			<td>JEM-1200EX</td>
			<td></td>
		</tr>
		<tr>
			<td>The Rotary Cell Culture System (RCCS)</td>
			<td>Synthecon</td>
			<td>RCCS-4HD</td>
			<td>3D culture</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>Optima XPN-100</td>
			<td>Exosome centrifuge</td>
		</tr>
	</tbody>
</table>

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.

Flow cytometry was used to identify the surface markers of SF-MSCs, according to the minimal criteria to define human MSCs recommended by the International Society for Cellular Therapy14,15. Flow cytometry analysis revealed that SF-MSCs cultured in this study met the identification criteria of MSCs. They were negative for CD34, CD45, and HLA-DR (below 3%) and positive for CD73, CD90, and CD105 (above 95%) (Figure 2).
<p class="jo...

Watch this Scientific Journal Video about Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture at JoVE.com

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

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. ...

We present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using our 3D bioreactor. These exosomes could be utilized in exosome biology research and clinical arthritis treatment. Demonstrating the procedure will be Dr.Yujie Liang.Start collecting the human mesenchymal cells, or MSCs, by centrifuging the synovial fluid at 1, 500 times G for 10 minutes at four degrees Celsius. After centrifugation, discard the supernatant and resuspend the cell pellet with 10 milliliters of PBS. Centrifuge the resuspended cells at 1, 500 times G for 10 minutes at four degree Celsius and discard PBS before resuspending the pellet with 10 milliliters of human MSC culture medium at a cell density of five times 10 to the fourth cells per milliliter and then plate the suspension in a 100-millimeter dish.Incubate the dish at 37 degree Celsius in an atmosphere containing 5%carbon dioxide. After two weeks, collect the cell culture supernatant to identify synovial fluid mesenchymal stem cells, or SF-MSCs, using flow cytometry. To do so, digest the third generation passage three of SF-MSCs by centrifugation at 1, 000 times G for five minutes at four degree Celsius.Then, discard the supernatant before collecting the cell pellet. Next, add 400 microliters of the blocking buffer to the cell pellet at the concentration of five times 10 to the fourth and allow the pellet to stand for 15 minutes at room temperature. After 15 minutes, centrifuge the cell suspension as described earlier, then resuspend the separated pellet in 100 microliters of 1X PBS.Add one microliter of the monoclonal fluorescent antibody at a dilution ratio of 1:100 per tube as described in the manuscript, and place the tube for incubation at room temperature for 30 minutes. Following incubation, wash the cells twice with 1X PBS and resuspend the cells in 100 microliters of 1X PBS. Then, detect the fluorophores in up to 10, 000 cells on a flow cytometer using red and FITC channels.To prepare the microcarriers, swell and hydrate dry 0.75 grams of the microcarriers in 1X DPBS at the concentration of 50 milligrams per gram for at least three hours at room temperature. Then, decant the supernatant to wash the microcarriers in fresh DPBS for five minutes. After replacing the medium with fresh 1X DPBS at 50 milligrams of the microcarriers per gram, sterilize the microcarriers by autoclaving at 121 degrees Celsius for 15 minutes at 15 pounds per square inch.Then, allow the sterilized microcarriers to settle before decanting the supernatant. Rinse the microcarriers in the culture medium at the concentration of 50 milligrams of the microcarriers per gram at room temperature. When the microcarriers are settled, discard the supernatant.To prepare perfusion bioreactor, sterilize the bioreactor by autoclaving as described earlier. When done, count the number of SF-MSCs to allocate 2.5 times 10 to the seventh SF-MSCs and microcarriers to the bioreactor perfused with 250 milliliters of the GMP-grade MSC culture medium. Place the bioreactor in an incubator with 5%carbon dioxide at 37 degrees Celsius at a speed of 15 rotations per minute.Change the culture medium every six days. After 14 days, collect the cell culture supernatants and microcarriers for further analysis. Centrifuge the cell culture supernatant at 300 times G for 10 minutes at four degrees Celsius and collect the supernatant while discarding the cellular debris.At four degree Celsius, sequentially centrifuge the supernatant at 2, 000 times G for 10 minutes and then at 10, 000 times G for 30 minutes to discard the larger vesicles and collect the pellet. After resuspending the pellet in 40 milliliters of PBS, centrifuge the cell suspension at 120, 000 times G for 70 minutes at four degree Celsius. Then, resuspend the collected pellet containing exosomes in 500 microliters of PBS.Dilute the freshly isolated exosome samples with sterile PBS to the concentration of 10 to the seventh to 10 to the ninth particles per milliliter and inject 500 microliters of the sample for each run into the nanoparticle tracking analysis, or NTA system, at 30 microliters per minute and 24.4 to 24.5 degrees Celsius. Manually set the capture and analysis system according to the manufacturer's protocol. Visualize the particles by laser light scattering and capture their Brownian motion on digital video.Next, analyze the recorded videotapes utilizing software, tracking at least 200 individual particles per run. For Western blotting, add 300 microliters of the lysis buffer and protease inhibitors cocktail to the exosomes with mixing by pipetting up and down. Then, allow the to stand on ice for 20 minutes.After centrifugation of the mixture at 9, 391 times G for 10 minutes at four degree Celsius, measure the protein concentration in the supernatant using a protein assay kit. Following the assay, heat the sample with 100 microliters of 4X protein loading buffer at 100 degree Celsius for 10 minutes. Next, load 15 microliters of proteins at a concentration of 10 milligrams per milliliter to run by gel electrophoresis at 120 volts for 70 minutes, and electro-blotting at 100 volts for 60 minutes at four degrees Celsius.Detect the non-exosome specific markers, such as calnexin and the exosomal biomarkers such as CD9, CD63, and CD81 by fluorescent Western blotting. In the study, flow cytometry was used to identify the surface markers of SF-MSCs. Flow cytometry analysis revealed that the cultured SF-MSCs were negative for CD34, CD45, and HLA-DR, and positive for CD73, CD90 and CD105, meeting the identification criteria of MSCs.Under inverted microscopy, it was noticed that the SF-MSCs proliferate on microcarriers. The SF-MSC proliferated in 3D culture more quickly from six days onwards as compared to 2D culture. The exosomes from SF-MSCs of 2D and 3D culture were identified using Western blotting and the exosomes were found to be expressing CD63, CD9, and CD81 while being negative for calnexin.The nanocyte analysis demonstrated that the diameter of 2D and 3D exosomes was approximately 120 nanometers. The transmission electron microscopic analysis revealed the morphology of 2D and 3D exosomes, showing roughly spheroidal vesicles. Using NTA analysis, the concentration of 30-to 160-nanometer sized particles was analyzed.After 3D culture, the particle concentration was 4.0 times 10 to the six per milliliter. However, after the 2D culture, the concentration of same-sized particles was 2.5 times 10 to the six per milliliter. Furthermore, 3D culture produced more exosome protein than 2D culture.The representative analysis shows the internalization of Dil-labeled exosomes by primary chondrocytes. Dil-labeled exosomes entered primary chondrocytes can be seen with a peak at three hour. In vitro delivery study demonstrated that exosomes could deliver sulfo-cyanine3-labeled microRNA-140 to chondrocytes.Plating the cells, starting the reaction in the bioreactor, and the of the supernatant are the most important steps in this protocol. 3D culture is commonly used for large-scale culture of adherent cells. Other methods such as spin flex can also be used for large-scale production of exosomes.Our method improves the yield of exosomes, thus enabling MSC exosome-based preclinical application.

large-scale-preparation-synovial-fluid-mesenchymal-stem-cell-derived

Research

JoVE Journal

Biology

3.8K visualizações.  The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital. Apresentamos um protocolo para produzir um grande número de exossomos de grau GMP a partir de células-tronco mesenquimais do líquido sinovial usando nosso biorreator 3D. Esses exossomos podem ser utilizados em pesquisas de biologia de exossomos e tratamento clínico da artrite. Demonstrando o procedimento será o Dr. Yujie Liang.Comece a coletar as células mesenquimais humanas, ou MSCs, centrifugando o líquido sinovial a 1, 500 vezes G por 10 minutos a quatro graus Celsius. Após ...