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