Name: Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture (Video) | JoVE Journal
Uploaded: 2022-07-26T12:30:00.000+00:00
Duration: 9 min 48 s
Description: Learn to produce GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor culture system. This protocol enables scalable manufacturing for arthritis research and therapeutic applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 into the articular cavity with a 7# needle.</li>
		<li>Extract 10 mL of the joint fluid. Cover the puncture site with the transfusion stick and press for 5 min.</li>
	</ol>
	</li>
	<li>Discard the SF supernatant after centrifugation at 1,500 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend the cell pellet with 10 mL of 1x phosphate buffer saline (PBS), centrifuge at 1,500 x g for 10 min at 4 &#176;C, and discard the PBS.</li>
	<li>Resuspend the pellet with 10 mL of human MSC culture medium at a cell density of 5 x 104 cells/mL (see Table of Materials), and then plate the suspension in a 100 mm dish.</li>
	<li>Incubate the dish at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>After 48 h, remove the non-adherent cells by changing the medium and wash with 1x PBS. Replace the medium every 3 days for 2 weeks.</li>
	<li>Collect the cell culture supernatants.</li>
	<li>Identify SF-MSCs using flow cytometry.
	<ol>
		<li>Digest the third generation (P3) of SF-MSCs, centrifuge at 1,000 x g for 5 min at 4 &#176;C. Discard the supernatant and collect the cell pellet. 
		NOTE: A passage is recognized as the sub-culture of the cells to another culture dish. The primary cells isolated from SF and seeded on dishes are labeled as passage zero (P0). At about 75% confluence, the cells are digested and detached from the dishes, seeded on other dishes, and labeled as P1.</li>
		<li>Add 400 &#181;L of blocking buffer (1% BSA in 1x PBS) to the cell pellet (5 x 104) and allow it to stand for 15 min at room temperature (RT).</li>
		<li>Centrifuge at 1,000 x g for 5 min at 4 &#176;C, discard the supernatant, and resuspend the pellet in 100 &#181;L of 1x PBS.</li>
		<li>Add 1 &#181;L of CD105, CD73, CD90, CD45, CD34, HLA-DR monoclonal fluorescent antibody (dilution ratio 1:100) (see Table of Materials) per tube and incubate at RT for 30 min.</li>
		<li>Wash twice with 1x PBS and discard the supernatant. Collect the cell pellet and resuspend in 100 &#181;L of 1x PBS.</li>
		<li>Detect on a flow cytometer up to 10,000 cells using filters of 525/50 and 585/40 to detect the fluorophores.</li>
	</ol>
	</li>
</ol>
2. 3D bioreactor cell culture
<ol>
	<li>Microcarrier preparation
	<ol>
		<li>Swell the dry 0.75 g of microcarriers (see Table of Materials) and hydrate in 1x Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (50 mL/g of microcarriers) for at least 3 h at RT.</li>
		<li>Decant the supernatant and wash the microcarriers for 5 min in fresh DPBS (50 mL/g of microcarriers). Discard the PBS and replace it with fresh 1x DPBS (50 mL/g of microcarriers).</li>
		<li>Sterilize the microcarriers by autoclaving (121 &#176;C, 15 min, 15 psi). Allow the sterilized microcarriers to settle, decant the supernatant.</li>
		<li>Briefly rinse the microcarriers in the culture medium (50 mL/g of microcarriers) at RT. Allow the microcarriers to settle, discard the supernatant.</li>
	</ol>
	</li>
	<li>Perfusion bioreactor
	<ol>
		<li>Sterilize the bioreactor by autoclaving (121 &#176;C, 15 min, 15 psi).</li>
		<li>Count the number of SF-MSCs and allocate 2.5 x 107 SF-MSCs and microcarriers to the bioreactor perfused with GMP-grade MSC culture medium (250 mL).</li>
		<li>Put the bioreactor in an incubator with 5% CO2 at 37 &#176;C. Rotate the bioreactor at a speed of 15 rpm. Change the culture medium every 6 days.</li>
		<li>Collect the cell culture supernatants and microcarriers for further analysis after culture for 14 days.</li>
	</ol>
	</li>
</ol>
3. Exosome identification and safety detection
<ol>
	<li>Ultracentrifugation
	<ol>
		<li>Centrifuge the cell culture supernatant at 300 x g for 10 min at 4 &#176;C and collect the supernatant; discard the cellular debris.</li>
		<li>Centrifuge the supernatants at 2,000 x g for 10 min at 4 &#176;C and collect the supernatant; discard the larger vesicles (apoptotic bodies and some larger microvesicles).</li>
		<li>Centrifuge the supernatant again at 10,000 x g for 30 min at 4 &#176;C to remove larger vesicles; collect the pellets and resuspend in 40 mL of 1x PBS.</li>
		<li>Centrifuge the resuspended pellets at 120,000 x g for 70 min at 4 &#176;C, discard the supernatant, and resuspend the pellets that contain exosomes in 500 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>Nanoparticle tracking analysis (NTA) 
	NOTE: For each run, 500 &#181;L of the sample were injected into the chamber at a flow rate of 30 &#181;L/min. Perform the analysis at 24.4 &#176;C-24.5 &#176;C.
	<ol>
		<li>Dilute the freshly isolated exosome samples with sterile 1x PBS to 1 mL containing 107-109 /mL of particles and inject them into the nanoparticle tracking analysis system (see Table of Materials).</li>
		<li>Manually set the capture and analysis system according to the manufacturer&#39;s protocol. Visualize the particles by laser light scattering and capture their Brownian motion on digital video.</li>
		<li>Analyze the recorded videotapes utilizing software (see Table of Materials) based on tracking at least 200 individual particles per run.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy
	<ol>
		<li>Fix the exosomes in 4% paraformaldehyde (in cold 1x DPBS) for 5 min.</li>
		<li>Mount 5 &#181;L of the exosomes on copper grids. In this experiment, the concentration of exosomes is 1 mg/mL quantified by a protein assay kit (see Table of Materials).</li>
		<li>Embed the exosomes in 3% phosphotungstic acid for 10 min on ice. Remove the excess acid and dry the samples at RT.</li>
		<li>Image the exosome samples by a TEM at an acceleration voltageof 100 kV.</li>
	</ol>
	</li>
	<li>Western blotting
	<ol>
		<li>Add 300 &#181;L of lysis buffer (1% Triton X-100, 0.1% SDS, 0.1 M Tris HCl, pH 7) and protease inhibitors cocktail (see Table of Materials) to the exosomes.</li>
		<li>Mix the exosomes in the lysis buffer by pipetting up and down and allow it to stand on ice for 20 min.</li>
		<li>Centrifuge the mixture at 9,391 x g for 10 min at 4 &#176;C and collect the supernatant. Measure the protein concentration using a protein assay kit (see Table of Materials).</li>
		<li>Add 100 &#181;L of 4x protein loading buffer and heat at 100 &#176;C for 10 min.</li>
		<li>Load 15 &#181;L of proteins at a concentration of 10 mg/mL and run by gel electrophoresis (120 V, 70 min) and electroblotting at 100 V, 60 min at 4 &#176;C.</li>
		<li>Detect the non-exosome-specific markers (calnexin) and exosomal biomarkers (CD9, CD63, and CD81) by fluorescent western blotting (see Table of Materials).</li>
	</ol>
	</li>
	<li>Safety test
	<ol>
		<li>For bacteria, fungi, and Mycobacterium tuberculosis detection follow steps 3.5.2-3.5.5.</li>
		<li>Seed 100 &#181;L of the exosome solution (1 mg/mL) on a blood agar culture plate (see Table of Materials) and incubate the culture plate at 37 &#176;C for 24 h.</li>
		<li>Seed 100 &#181;L of the exosome solution (1 mg/mL) on a sabouraud agar medium plate (see Table of Materials) and incubate at 35 &#176;C for 48 h.</li>
		<li>Seed 100 &#181;L of the exosome solution (1 mg/mL) on a Lowenstein-Jensen culture medium plate (see Table of Materials) and incubate at 37 &#176;C for 3 weeks.</li>
		<li>Detect the appearance of bacteria, fungi, and Mycobacterium tuberculosis colonies by macroscopic observation. 
		NOTE: Criteria for evaluating the safety of exosomes are the absence of microorganisms on the culture plate.</li>
		<li>For Mycoplasma detection, follow steps 3.5.7-3.5.9.</li>
		<li>Resuspend the exosomes in 50 &#181;L of buffer solution included in the kit and heat at 95 &#176;C for 3 min.</li>
		<li>Amplify the Mycoplasma in exosome solution using a PCR Mycoplasma detection kit (see Table of Materials).</li>
		<li>Detect the PCR products on 1.5% agarose gel electrophoresis (30 min, 120 V).</li>
	</ol>
	</li>
</ol>
4. In vitro exosome function detection
<ol>
	<li>Exosome labeling
	<ol>
		<li>Label 100 &#181;L of exosomes (1 mg/mL) with 1 mM Dil (1000x) (see Table of Materials) and incubate at RT for 30 min.</li>
		<li>Recover the exosomes by ultracentrifugation at 100,000 x g for 70 min. Resuspend the pellet in 500 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>Loading of Cy3-miR-140 mimics into exosomes
	<ol>
		<li>Mix 1 &#181;g/&#181;L of exosomal protein and 5 &#181;mol/mL of Cy3-miR-140 in a final volume of 400 &#181;L of electroporation buffer (1.15 mM potassium phosphate (pH 7.2), 25 mM potassium chloride and 21% (v/v) (see Table of Materials).</li>
		<li>Transfer the mixture into cold 0.4 cm electroporation cuvettes and electroporate at 300 V/100 &#181;F capacitance using a gene transfection system (see Table of Materials).</li>
		<li>Immediately after electroporation, maintain the mixture at RT for 30 min to ensure the exosome membrane is fully restored.</li>
		<li>Treat the mixtures with one unit of RNase A (see Table of Materials) for 20 min to eliminate the miRNA mimics outside the exosomes.</li>
		<li>Ultracentrifugate the exosome mixture at 120,000 x g for 90 min, discard the supernatant and resuspend the exosomes in 500 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>Exosome uptake assay
	<ol>
		<li>Seed the chondrocytes (1 x 107) in 35 mm confocal dishes with 1 mL of DMEM/F12 containing 10% FBS.</li>
		<li>After 24 h, treat the chondrocytes with DiI-labeled exosomes (100 ng/mL) or cy3-miR-140 loaded exosomes (100 ng/mL) for 1 h.</li>
		<li>Wash with 1x PBS three times, and then label with DAPI (10 ng/mL) for 10 min at RT.</li>
		<li>Record the fluorescence signal in confocal laser scanning fluorescence microscopy (CLSM) using the appropriate filters (see Table of Materials).</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform statistical analysis using appropriate statistical software. 
	NOTE: Representative data in each figure were expressed as the mean &#177; SD. The Student&#39;s t-test was used for the comparison of two groups using statistics software. A one-way ANOVA followed by Tukey&#39;s multiple was performed in the case of comparisons among multiple groups. P values &#60;0.05 (*), &#60;0.01 (**), or &#60;0.001 (***).</li>
</ol>

<ol><li>Cross, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+global+burden+of+hip+and+knee+osteoarthritis%3A+estimates+from+the+global+burden+of+disease+2010+study%5BTitle%5D)+AND+%22Annals+of+the+Rheumatic+Diseases%22%5BJournal%5D)">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/pubmed?term=((Osteoarthritis%3A+a+disease+of+the+joint+as+an+organ%5BTitle%5D)+AND+%22Arthritis+%26+Rheumatology%22%5BJournal%5D)">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis & 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/pubmed?term=((Unlike+bone%2C+cartilage+regeneration+remains+elusive%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Increased+recruitment+of+endogenous+stem+cells+and+chondrogenic+differentiation+by+a+composite+scaffold+containing+bone+marrow+homing+peptide+for+cartilage+regeneration%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">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/pubmed?term=((Survival+analysis+of+revision+autologous+chondrocyte+implantation+for+failed+ACI%5BTitle%5D)+AND+%22American+Journal+of+Sports+Medicine%22%5BJournal%5D)">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/pubmed?term=((Autologous+chondrocyte+implantation%3A+past%2C+present%2C+and+future%5BTitle%5D)+AND+%22Sports+Medicine+and+Arthroscopy+Review%22%5BJournal%5D)">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/pubmed?term=((Native+joint-resident+mesenchymal+stem+cells+for+cartilage+repair+in+osteoarthritis%5BTitle%5D)+AND+%22Nature+Reviews+Rheumatology%22%5BJournal%5D)">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/pubmed?term=((Intra-articular+injection+of+mesenchymal+stem+cells+for+the+treatment+of+osteoarthritis+of+the+knee%3A+a+proof-of-concept+clinical+trial%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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., Noël, D., Jorgensen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+for+the+management+of+inflammation+in+osteoarthritis%3A+state+of+the+art+and+perspectives%5BTitle%5D)+AND+%22Osteoarthritis+Cartilage%22%5BJournal%5D)">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/pubmed?term=((In+vitro+and+in+vivo+potentialities+for+cartilage+repair+from+human+advanced+knee+osteoarthritis+synovial+fluid-derived+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">In vitro and in vivo potentialities for cartilage repair from human advanced knee osteoarthritis synovial fluid-derived mesenchymal stem cells.</a> Stem Cell Research & Therapy. 9 (1), 329(2018).</li>
<li>Jia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic-activated+cell+sorting+strategies+to+isolate+and+purify+synovial+fluid-derived+mesenchymal+stem+cells+from+a+rabbit+model%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((Concise+review%3A+MSC-derived+exosomes+for+cell-free+therapy%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Engineering+mesenchymal+stem+cells+to+improve+their+exosome+efficacy+and+yield+for+cell-free+therapy%5BTitle%5D)+AND+%22Journal+of+Extracellular+Vesicles%22%5BJournal%5D)">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/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+international+society+for+cellular+therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">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/pubmed?term=((Concise+review%3A+the+surface+markers+and+identity+of+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Concise+review%3A+Multifaceted+characterization+of+human+mesenchymal+stem+cells+for+use+in+regenerative+medicine%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((Mesenchymal+stem+cells+for+regenerative+medicine%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Exosomes+derived+from+human+neural+stem+cells+stimulated+by+interferon+gamma+improve+therapeutic+ability+in+ischemic+stroke+model%5BTitle%5D)+AND+%22Journal+of+Advanced+Research%22%5BJournal%5D)">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/pubmed?term=((Migration+ability+and+Toll-like+receptor+expression+of+human+mesenchymal+stem+cells+improves+significantly+after+three-dimensional+culture%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">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/pubmed?term=((The+influence+of+spheroid+formation+of+human+adipose-derived+stem+cells+on+chitosan+films+on+stemness+and+differentiation+capabilities%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Epigenetic+changes+of+mesenchymal+stem+cells+in+three-dimensional+%283D%29+spheroids%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Neurochemistry+International%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+culture+of+MSCs+produces+exosomes+with+improved+yield+and+enhanced+therapeutic+efficacy+for+cisplatin-induced+acute+kidney+injury%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury.</a> Stem Cell Research & Therapy. 11 (1), 206(2020).</li>
</ol>

The authors declare that they have no competing financial interests.

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><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>&amp;nbsp;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>&amp;nbsp;A5270</td><td>Western blotting</td></tr><tr><td>CD9 antibody</td><td>Abclonal</td><td>&amp;nbsp;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

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...

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. ...

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

Research

JoVE Journal

Biology

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.

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

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.

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

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

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.

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

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Preparation of Exosomes for siRNA Delivery to Cancer Cells

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

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

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

Large-Scale Screens of Metagenomic Libraries

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.

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

Learn to produce GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor culture system. This protocol enables scalable manufacturing for arthritis research and therapeutic applications.

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

Explore More Videos