Medicine
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Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs
Chapters
Summary July 2nd, 2016
A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.
Transcript
The overall goal of this procedure is the non-invasive collection of synovial fluid and bone marrow from minipigs to collect mesenchymal stem cells to be used in regeneration and immunomodulation studies. This method can help answer key questions in the otologist MSC therapy field, such as how to establish the synovial fluid in MSC, and how to characterize them in comparison to bone marrow MSC. The main advantage of this technique is to effectively establish the key properties of synovial fluid and bone marrow-derived MSCs from minipigs, which will be useful for application in patients.
The implications of this technique extend toward therapy of various joint diseases, because the population of the synovial fluid-derived MSC in the synovial cavity is significantly increased in athletic patients. Though this method can provide insight into regenerative medicine, it can also be applied to other studies such as autoimmune disease research. Because minipig MSCs are autologous, immunosuppression might interest humans.
In addition to the other scientists, demonstrating the procedure will be Hyeon-Jeong Lee and Won-Jae Lee from my level three. To begin, orient an anesthetized minipig to access the iliac crest. Next, depilate a 15-square-centimeter region on the iliac crest, using a razor or clippers.
Then, alternately scrub the exposed skin three times with povidone-iodine and 70%ethanol. Once completely dried, apply sterile drapes. Now, pre-coat a ten-milliliter syringe with heparin, using a three-to four-milliliter rinse.
This will prevent the bone marrow from coagulating while it is extracted. Then, precisely at the iliac bone crest, tightly attach a bone marrow extractor to the heparin-coated syringe, and extract at least five milliliters of bone marrow. To isolate the cells from the extracted bone marrow, first dilute it with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.
Then, load three milliliters of density centrifugation media into a second 15-milliliter conical tube. Next, carefully transfer four milliliters of the diluted bone marrow onto the density centrifugation media to create two-solution layers. Now, centrifuge the tube at 400 Gs for 40 minutes at room temperature.
The desired MNCs are collected in the central white layer. Aspirate just this layer. Eject the MNC layer into a 15-milliliter conical tube, and dilute it with seven to eight milliliters of DPBS.
Then, centrifuge the cells at 500 Gs for ten minutes at room temperature. Discard the supernatant, and wash the palleted cells twice with ten milliliters of DPBS, repeating the previous centrifugation cycle. After the two washes, we suspend the MNCs in two milliliters of ADMEM.
Then, load the cells onto a 35-millimeter tissue culture dish, and incubate them at 38.5 degrees Celsius in a humidified 5%carbon dioxide incubator. After 48 hours, aspirate the medium along with the unattached MNCs. Then, add back two milliliters of fresh ADMEM, and continue the incubation, changing the medium every three days.
When the cells reach 70 to 80%confluency, wash the culture with fresh DPBS. Then, dissociate the cells, using one milliliter of 0.25%Trypsin-EDTA for three to five minutes at 37 degrees Celsius. Collect the cells in ten milliliters of ADMEM, and transfer them into a 15-milliliter conical tube.
Then, spin the cells down at 300 Gs for five minutes at room temperature, and discard the supernatant. Now, suspend the harvested MNCs in one milliliter of fresh ADMEM, and measure their density. Finish with plating between 1/2 and three-quarter million cells into T25 flasks with four milliliters of ADMEM.
Incubate the flasks just as the plates, and change the medium every third day. Prepare a 15x10 centimeter section of skin over the femoral joint, as was done when preparing for bone marrow collections. Then, palpate for the patella and tibia crests below the skin to identify the synovial joint, and insert a three-milliliter syringe with a 23-gauge needle, precisely into the joint.
Using gentle suction, extract between 1/2 and one milliliter of fluid, and immediately withdraw the needle. If the synovial fluid volume is too small, then flush the cavity with DPBS to collect synovial fluid with DPBS. The volume of DPBS to use varies with the size of the synovial cavity.
The optimal volume of synovial fluid is limited in healthy joint. The volume varies with the size and pathological condition of its joint. To isolate the cells from the synovial fluid, dilute the fluid with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.
Next, we move the debris by filtering the dilution through a 40-micron nylon cell strainer. Then, add ten more milliliters of DPBS, and centrifuge the suspension at 400 Gs for ten minutes at room temperature. Next, discard the supernatant, and wash the cells two times, using DPBS.
We suspend the washed cell pallet in two milliliters of ADMEM. Then, proceed with culturing the cells as was demonstrated with the MSCs. MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro.
The morphology of synovial fluid-derived MSCs is similar to that of bone marrow-derived MSCs:both have a fibroblastic spindle shape and are homogeneously adherent. The MSCs showed positive expression for alkaline-phosphatase staining after the third subculture was completed. Next, surface markers were analyzed in both cultures.
As expected, both types of MSCs significantly expressed CD29, CD44, and Vimentin. By contrast, expression of CD34 and CD45 were negative and low. These results were identical to those obtained through distinctive MSCs, including cells isolated from synovial fluid.
After watching this video, you should have a good understanding of how to non-invasively aspirate synovial fluid and bone marrow in order to isolate their MSC from minipigs and how to perform the comparison and phenotyping of this MSC. While attempting this procedure, it's important to remember to locate the objective point on the iliac crest and the synovial, determined by careful palpation. Following this procedure, other methods like in models of osteoarthritis or rheumatoid arthritis can be performed in order the answer additional questions like whether not only from marrow MSC but also synovial fluid MSCs have a same kind capacity for cartilage or bone initiation and immunosuppressions.
After its development, this technique advanced autologous therapy of joint diseases by helping to characterize MSCs derived from attacked or inflamed joint in arthritis patients.
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