Name: large-scale preparation of synovial fluid mesenchymal stem cell-derived exosomes by 3d bioreactor culture
Uploaded: 2022-07-26T12:30:00
Description: Watch this Scientific Journal Video about Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture at JoVE.com

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. 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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. 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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

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...