This method can help answer key questions in the sorting protocol in the stem cell field by providing a procedure for purifying mesenchymal stem cells directly from synovial fluid. The main advantage of this technique is that we have establish a simple and economic protocol for the isolation and the purification of MACS from rabbit synovial fluid. Generally in the research, due to this method, we make great efforts, because plurating of cell population is dependent on producing a single cell suspension.
We first had the idea for this method when we noticed that only the magnetic-activated cell sorting method is currently approved by the FDA for clinical applications. Begin this procedure with animal anesthesia as described in the text protocol. To perform collection of articular synovial fluid from the knee of the rabbit, select an area about five by five centimeters in size around the knee.
Shave the rabbit hair from around this area using a safety electric shaver. Alternately, disinfect the procedure site with povidone iodine solution and 75%ethanol three times. Then, apply sterile drapes after area has thoroughly dried.
Use a sterile hypodermic syringe to inject one to two milliliters of isotonic saline solution into the knee joint cavity from the lateral articular space. Move the knee three to four times, and then aspirate all the synovial fluid at room temperature. Filter the synovial fluid through a 40 micron nylon cell strainer to remove any debris, within four hours.
To culture the rabbit SF-MSCs, collect the filtered fluid in 50 milliliter centrifuge tubes, and centrifuge at 1, 500 rpm for ten minutes at room temperature. Discard the supernatant after the centrifugation and wash the pellet with PBS. After resuspending the pellet with the complete culture medium, plate the medium in 100 milliliter dishes.
Incubate the dishes at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide. After 48 hours, replenish the dishes with fresh medium to remove any non-adherent cells. Replace the medium twice a week for two weeks as passage zero.
After 14 days following the initial plating, many colonies should've formed in the culture dishes. Select the colonies larger than two millimeters in diameter. Mark where the selected colonies are located, by tracing their circumference on the dish bottom.
Discard the colonies less than two millimeters wide using cell scrapers. Digest the selected colonies with about five microliters of 0.25%trypsin using a cloning cylinder, and transfer the colonies to a new dish to passage. When the cells reach about 80%confluency, aspirate the medium.
Then, add one to two milliliters of 0.25%trypsin EDTA to each dish. Incubate the dishes for two to three minutes to allow cell detachment. Once the cells are detached, add an equal amount of the culture medium to inactive the trypsin.
Pass the cell suspension through a 40 micron cell strainer and collect the filtrate in a 15 milliliter tube. Then spin down the cells at 600 times G for ten minutes at room temperature. Resuspend the cell pellet in MACS Running Buffer.
To perform magnetic labeling, determine the cell number using a hemocytometer. Centrifuge the cell suspension at 300 times G for ten minutes at four degrees Celsius. After completely aspirating the supernatant, add 80 microliters of resuspension buffer per ten million total cells.
For ten million total cells, add 20 microliters of microbeads conjugated with a monoclonal anti-rabbit CD90 antibody. Mix the magnetic beads and cells evenly in the tubes. Then incubate them at four degrees Celsius for 15 minutes in the dark.
Add one milliliter of buffer per ten million cells to the tube, and then centrifuge at 300 times G for ten minutes at four degrees Celsius, to wash the cells. Discard the supernatant after centrifugation. Resuspend the pellet in 500 microliters of buffer per ten million cells.
Place the column, with the column wings to the front, into the magnetic field of the magnetic separator. Rinse the magnetic separator column with 500 microliters of the buffer per ten million cells. Transfer the single cell suspension into the column.
Let the negative cells pass through the magnetic field to discard these unlabeled cells. Wash the column one to two times with 500 microliter of the buffer per ten million cells, and discard the flow through. Transfer the column into a 15 milliliter centrifuge tube.
Add one milliliter of the buffer per ten million cells to the column. Then immediately push the plunger into the column, to flush out the magnetically labeled cells. Repeat the aforementioned procedure with a second magnetic separator column, to increase the purity of CD90-positive cells, which can enrich the eluted fraction.
After centrifuging the cell suspension at 300 times G for ten minutes, aspirate the supernatant, and resuspend the pellet with the culture medium. Inoculate the cells in 100 milliliter dishes after the magnetic-activated cell sorting. Incubate the dishes at 37 Celsius in a humidified cell incubator with 5%carbon dioxide.
When 80 to 90%confluence of the primary culture is achieved, at about seven to ten days, digest the adherent cells with 0.25%trypsin EDTA and passage them at a one to two dilution to make passage two. Use the same method to pass the cells to passage three, which can be used for the in-vitro assays. Shown here, is the morphology of the monolayer cultured rabbit SF-MSCs before and after MACS, under an inverted microscope.
Before sorting, the adherent rabbit synovial fluid cell populations display heterogeneity. They contain diverse cell types and sizes such as oval, long-spindle, short-spindle, and stellate morphology. The main morphology of passage two and passage six, is a spindle morphology.
Following MACS with CD90, the cell populations exhibit a homogenous morphology. With the sub culture, the cell number increases. Flow cytometry analysis demonstrated that rabbit SF-MSC cells were positive for the MSC markers CD44 and CD105.
While negative for the endothelial cell marker CD34, and for the hematopoietic cell marker CD45. The results showed that before MACS, the rabbit SF-MSC population was composed of approximately 40%MSC cells. After MACS, the enriched population contained greater than 99%MSC cells.
Shown here, is flow cytometry analysis of the CD90-positive marker before and after MAC sorting. Alizarin Red staining demonstrated that mineralized nodules formed under osteogenic induction for three weeks. After three weeks of adipogenic induction, accumulation of lipid rich vacuoles was detected by intracellular Oil Red O staining.
After tree weeks of chondrogenic induction, the cell pellet was histologically assayed with Toluidine Blue staining. The positive acidic proteoglycan characterized the chondrocyte-like cells. After three weeks of induction, the relative mRNA expression of the osteoblast marker, the adipogenic marker and the chondrogenic marker was detected.
Once mastered, this technique can be done in two to four hours. After watching this video, you should have a good understanding of the straight forward isolation and the purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid. While attempting this procedure it's important to remember to select those colonies larger than two milliliters in diameter.
Also produce a single cell suspension before immunomagnetic sorting, as unwanted cells attached to a labeled cell will remain on the magnetic bead. Following this procedure, other methods like fluorescence activated cell sorting can be performed in order to answer additional questions addressing the safety and efficacy of purification of mesenchymal stem cells. After it's development, this technique paved the way for researchers in the field of articular cartilage regeneration to explore the ideal CD cells in articular cartilage repair.
Don't forget that the operations were performed on arch cooling bench, and the precautions such as gloves, should be always be taken while performing this procedure. The implications of this technique extend towards therapy of articular cartilage injury, because synovial fluid derived MASC are promising candidates for articular cartilage regeneration.
