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>

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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 derived from MSCs have been used for therapeutic interventions, which have attracted the most attention in OA therapy.
Currently, there are two methods to propagate MSCs, 2D culture and three-dimensional (3D) culture19. 2D culture is a conventional way to culture MSCs for in vitro studies with low costs. However, it is time-consuming and limited in scale potential. Also, MSC propagation in the 2D microenvironment quickly deduces the stemness20,21, one of the most critical characteristics of MSCs. Thus, 2D culture cannot fulfill the requirements for MSC therapy. In this study, to the aim of SF-MSCs in OA therapy, we endeavor to maintain SF-MSC characteristics using the culture of a 3D bioreactor, which can more accurately mimic the biological microenvironment. Recently, other researchers have used scaffolds or microcarrier-based 3D to culture MSCs. We found that the 3D collagen scaffolds allowed more concentrated exosomes produced by human bone marrow-derived MSCs than 2D culture23.
As exosomes can perform a large part of MSC function while avoiding some shortcomings of MSCs, we aim to produce exosomes for OA therapy. This study further detected the exosome production and delivery function using this system based on the large quantity and high quality of MSC propagation of 3D culture. The Rotary Cell Culture System (RCCS) was used for 3D culture to produce exosomes from large-scale MSC propagation. Compared to traditional 2D flask culture, this 3D culture system may avoid contamination from repeated medium change and cell passage. More importantly, this study showed that a 3D bioreactor could enhance exosome production to meet the clinical study requirements. Of note, exosomes isolated from 3D culture supernatants can deliver miRNAs into cells, suggesting that 3D culture does not interfere with exosome function. Microbial and endotoxin detection results further support that this study has established a protocol that could produce exosomes, which are biologically safe and promising for OA therapy. At present, the exosome quantity produced in this study cannot satisfy commercial needs. Hence, strategies for an enormous amount of exosome production need to be developed.

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).
Under inverted microscopy, it was noticed that the SF-MSCs proliferate on microcarriers (Figure 3A). After cells were digested and washed from the 2D culture plate and 3D culture microcarriers, the cell number was counted. Compared to 2D culture, 3D culture induced the SF-MSC to proliferate more quickly from 6 days onwards (Figure 3B). Results of three independent experiments were presented.
To identify the exosomes (Exos) from SF-MSCs of 2D and 3D culture, the exosome-associated proteins (CD63, CD9, and CD81) and negative protein (calnexin) were detected using western blotting. Results revealed that the 2D-Exos and 3D-Exos express CD63, CD9, and CD81, while they are negative for calnexin (Figure 4A). Also, the exosome diameter and morphology were assayed using NTA and TEM. Nanosight analysis demonstrated that the diameter of 2D-Exos (Figure 4B) and 3D-Exos (Figure 4D) is approximately 120 nm. Transmission electronic microscope analysis revealed the morphology of 2D-Exos and 3D-Exos, showing roughly spheroidal vesicles (Figure 4C,E).
After 3D culture, the particle concentration of 30-160 nm sized particles is 4.0 x 106 per mL analyzed using NTA. However, after the 2D culture, the particle concentration of 30-160 nm particles is 2.5 x 106 per mL (Figure 5A). When calculating the exosome protein yield in the medium, 3D culture produced more exosome protein than 2D culture (Figure 5B). Thus, compared to 2D culture, 3D culture significantly enhanced exosome yield.
The exosomes were first labeled with Dil, and then incubated at a concentration of 10 &#181;g/mL with the chondrocytes for 1 h, 2 h, and 3 h to examine whether exosomes can enter primary chondrocytes in vitro. Dil-labeled exosomes entered primary chondrocytes, with the peak seen at 3 h (Figure 6).
To detect the function of exosomes as nanocarriers, exosomes were loaded with Cy3-labeled miR-140 through electroporation, and then treated with chondrocytes at a concentration of 10 &#181;g/mL for 3 h. Results demonstrated that exosomes could deliver miR-140 to chondrocytes (Figure 7). All the results met the specifications; all samples were sterile and negative for Mycoplasma.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62221/62221fig01.jpg" /> 
Figure 1: Schematic diagram of exosomes isolated from hSF-MSCs in vitro. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62221/62221fig02.jpg" /> 
Figure 2: Identification of SF-MSCs by flow cytometry. Flow cytometry shows the positive or negative immunophenotype of hSF-MSCs. (A) Labeling with an IgG1 isotype control antibody. (B) CD73 is positive, and CD34 is negative. (C) CD90 is positive, and CD45 is negative. (D) CD105 is positive, and HLA-DR is negative, known as MSC markers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62221/62221fig03.jpg" /> 
Figure 3: SF-MSC growth curve. (A) Representative images showing SF-MSCs (red arrows) on microcarriers under inverted microscopy (scale bar =100 &#181;m). (B) The growth curve of SF-MSC under 2D and 3D culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62221/62221fig04.jpg" /> 
Figure 4: Identification of exosomes. (A) Western blotting results of the 2D-Exos and 3D-Exos. (B) Nanosight analysis of the diameter of 2D-Exos and 3D-Exos. (C) TEM detection of the morphology of 2D-Exos and 3D-Exos. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62221/62221fig05.jpg" /> 
Figure 5: The enhanced yield of exosome production by 3D bioreactor culture. (A) Representative results of exosome size analyzed by NTA. (B) Protein yield = exosomal protein (&#181;g)/conditioned medium (mL). Plots show yield for each method and the mean &#177; SD of all measurements (** p &#60; 0.01). Statistical comparisons were performed by one-way ANOVA with post-hoc Bonferroni&#39;s correction and by Student&#39;s t-test. ** p &#60; 0.01 was considered to be a significant difference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62221/62221fig06.jpg" /> 
Figure 6 Representative images showing the internalization of Dil-labeled exosomes by primary chondrocytes. Chondrocytes were incubated with Dil-labeled exosomes for 1 h, 2 h, and 3 h. Exosomes were labeled with Dil (red), and nuclei were labeled with Hoechst (blue). Samples were detected at 60x magnification. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62221/62221fig07.jpg" /> 
Figure 7: In vitro delivery of miR-140 by MSC exosomes. After up-taking Cy3-labeled miR-140 that was encapsulated in SF-MSCs-derived exosomes, chondrocytes were imaged. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62221/62221fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

4.1K Views.  The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital. Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

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

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 ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...