The overall goal of the following experiment is to grow human articular chondrocytes in synovial fluid in long-term cultures. This is achieved by first encapsulating chondrocytes in alginate beads. Then the chondrocytes are cultured in synovial fluid, which allows the chondrocytes to proliferate in a 3D environment that mimics their normal growing conditions.
The median must be changed periodically to allow the cells sufficient access to nutrients. The final step of the procedure is to harvest the chondrocytes from the alginate beads for gene expression and histological analysis. These analyses confirm that chondrocytes cultured in alginate beads are viable, proliferating, and express a significant amount of cartilage matrix.
The main advantages of using this technique over existing methods is that we're analyzing the cartilage cells in three dimensional settings instead of 2D. And also we're using synovial fluid as the media instead of artificial media. So the combination of using three dimensional construct and using synovial fluid allows us to analyze them in a more natural environment as opposed to the two dimensional setting on a plate and the artificial media.
The method can help answer key questions of the field of cartilage biology and tissue engineering. For instance, how do cartilage cells or chondrocytes behave in synovial fluid over long term? And also, should we accustom the cartilage cells to synovial fluid before transplanting into the hosts?
Prepare a one milliliter vial of human articular chondrocytes or hacks by thawing them in warm water for a minute. Then mix the hacks into a vial containing two milliliters of warmed chondrocyte growth media. To remove the freezing media, which contains DMSO pellet the cells at 3000 RPM for three to five minutes, discard the supernatant and resuspend the cells in seven milliliters of chondrocyte growth media.
Next, transfer the cells to a 10 centimeter plate and culture them until the cells are confluent. Hacks should be used at a passage number no higher than three or four. Once confluent, wash the hacks with a sodium chloride solution rather than PBS.
Then collect cells by tryps using 0.5 milliliters of standard tripsin EDTA solution. Once the cells have detached, spin them down and they will be ready for 3D encapsulation. Picking up with app pelleted hacks, remove the supernatant and then suspend them in a 1.2%alginate solution at a density of 800, 000 cells per milliliter.
Now prepare a calcium chloride solution at five times the volume of hack alginate solution. Keep the solution stirring slowly while the hack alginate is being prepared. Pipette the hack alginate solution into a 12 milliliter syringe attached to a 22 gauge needle.
When the calcium chloride is ready, hold the syringe tip about six inches above the surface of the calcium chloride solution. The height of the syringe is over. The calcium chloride solution is really important.
If you don't hold it high enough, the beads won't be spherical and shaped. Then add the hack alginate mixture.Dropwise. Typically, the result in beads are two millimeters in diameter with about 10 to the fourth cells per bead.
To enhance the integrity of the beads, allow the beads to stir in solution for 20 to 25 minutes. Once the beads have finished stirring, allow them to settle and decant off the calcium chloride solution. Next, wash the beads two to three times with sodium chloride solution at about two to three times the volume of the beads.
Instead of using filters, keep the beads moist by allowing them to settle between wash solution changes. Follow the calcium chloride washes with one wash and chondrocyte differentiation medium. Then using a spatula, transfer the beads to culture dishes culture the beads for 48 hours and chondrocyte growth medium before switching them to chondrocyte.
Differentiation medium with synovial fluid, synovial fluid must be prepared just after it is harvested. Transfer fresh synovial fluid to 15 milliliter tubes and immediately centrifuge them at 3000 RPM for 15 minutes. To remove cell debris, the supernatant is clean, synovial fluid, aliquot the fluid into 1.5 milliliter micro centrifuge tubes to be stored at minus 80 degrees Celsius until an optimal concentration of synovial fluid is determined.
Use different culture media composed of synovial fluid and chondrocyte differentiation, medium at varying volumetric ratios with a constant concentration of ascorbic acid and of calcium chloride culture. The plates on a rocking platform in a 37 degrees Celsius incubator. Change the media every two to three days before removing old media.
Dilute it with calcium chloride solution to reduce its viscosity and coincidentally strengthen bead integrity. Then slowly pipette it from the plates. The beads can be cultured for up to four weeks before harvesting, depending on their proliferation matrix production and the goals of the experiment.
When harvesting the hacks from the alginate beads for gene expression or histological analysis, wash the alginate beads three times in calcium chloride solution without using a filter for gene expression analysis. Isolate the chondrocytes by immersing them in sodium citrate at a volume four to five times that of the beads. Agitate the mixture for 20 to 30 minutes, then spin down the cells in 15 milliliter tubes.
Discard the supernatant and resuspend the cell pellet and cell lysis buffer from a conventional kit, and proceed with the standardized RNA purification protocol to prepare the washed beads for fixation. Follow the calcium chloride washes with an overnight Wash Inc. Chondro differentiation medium.
Fix the beads in two milliliters of 70%ethanol for greater than 15 minutes at four degrees Celsius and proceed with histological analysis for dappy staining. Incubate the alginate beads in DAPI solution for an hour under gentle rocking. Then wash the beads three times with calcium chloride solution before visualizing the alginate beads under a fluorescent microscope.
The beads can also be sectioned for Ian Blue and h and e staining histological sectioning of dappy stained alginate beads overlaid with brightfield images showed that there was uniform distribution of the cells within beads cultured under all of the conditions from media lacking synovial fluid to media based entirely on synovial fluid. At the end of the 21 day culture period, gene expression was measured from human articular chondrocytes, cultured in synovial fluid, pooled from six patients with osteoarthritis consistent with previous reports. Day zero chondrocytes expressed minimal levels of cartilage matrix genes 3D culturing of chondrocytes with chondrocyte differentiation, medium or medium supplemented with synovial fluid significantly increased cartilage gene expression of collagen two acan and MMP 13, which indicates chondrocyte red differentiation.
By day 21, increasing the percentage of synovial fluid in the media resulted in comparable levels of cartilage matrix markers. Collagen two, ANDCAN mRNA expression chondrocytes cultured in 100%synovial fluid even exhibited a decrease in cartilage degrading enzyme MMP 13 mRNA expression as compared to those cultured in medium alone. Interestingly, the expression level of cell death indicator caspase three gradually decreased with increasing ratios of synovial fluid, suggesting that synovial fluid culturing has led to decreased apoptosis levels.
Thus culturing human articular chondrocytes and high levels of synovial fluid in a 3D setting is a feasible technology. Once mastered, the encapsulation technique could take less than an hour to complete. You want to make sure to wash the beads thoroughly between media changes.
And also you want to be careful not to damage the beads while using the pipette.
