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 ...

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

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).
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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

Research

JoVE Journal

Biology

4.1K Views.  The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital. We presenteren een protocol om een groot aantal GMP-grade exosomen te produceren uit synoviale vloeistof mesenchymale stamcellen met behulp van onze 3D-bioreactor. Deze exosomen kunnen worden gebruikt in exosoombiologisch onderzoek en klinische artritisbehandeling. De procedure wordt gedemonstreerd door Dr.Yujie Liang.Begin met het verzamelen van de menselijke mesenchymale cellen, of MSC's, door de synoviale vloeistof te centrifugeren op 1, 500 keer G gedurende 10 minuten bij vier grad...

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

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

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.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Cancer Research

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

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

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

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.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Developmental Biology

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

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

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.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioengineering

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

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 ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

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

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

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.

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

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 ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

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

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