The overall goal of this procedure is to induce chondrogenic differentiation of ASC's for defective cartilage repair. This method is to induce the prechondrogenic differentiation of ASC's using centrifugal gravity force. Though this method can provide insight into chondrogenic differentiation of ASC's, it can also be applied to other systems, such as osteogenic and fibrogenic differentiation.
The main advantage of this technique is that chondrogenic differentiation can be easily induced from stem cells without treating any cytokines. Demonstrating the procedure will be Yeri Alice Rim and Yoojun Nam from my laboratory. Begin by harvesting 80%confluent adipose-derived stem cells from a 100 milliliter culture dish by discarding the medium and washing with five milliliters of PBS.
Then, add one milliliter of PBS containing one millimolar EDTA, and incubate for two minutes at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide. After two minutes, tap the culture plate gently then add four milliliters of fresh medium and transfer the cells to a 15 milliliter conical tube. Centrifuge the cells at 250 times g for two minutes at room temperature.
After centrifugation, remove the supernatant without disturbing the pellet. Then, re-suspend the pellet in 10 milliliters of medium. Count the cells using a hemocytometer.
For centrifugal gravity loading, transfer 2.5 times ten to the fifth cells to a new 15 milliliter conical tube, and centrifuge at 2400 times g for 15 minutes. Following the centrifugation, proceed to either pellet or micromass culture. Immediately after centrifugation, aspirate the supernatant and at 500 microliters of chondrogenic differentiation medium, or CDM, and 50 micrograms per milliliter of freshly-prepared L-ascorbic-acid 2-phosphate to the pellet.
A positive control, induce chondrogenic differentiation and collect its cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1. Loosely cap the tubes and incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide for three weeks. Immediately after the gravity-loading centrifugation, aspirate the supernatant and re-suspend the pellet in 10 microliters of CDM.
To form a micromass, pipette the re-suspended cells into the center of a well of a 24 well plate and then place the plate into the incubator. After two hours, carefully add 500 microliters of CDM to the well, pipetting against the wall of the plate to avoid disrupting the micromass. As a positive control, perform a micromass culture with uncentrifuged cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1.
Incubate the micromass for three weeks. On day 21 after micromass formation, use a pipette to harvest the spheroid pellets, and then wash with 1X PBS. Fix the spheroid pellets by immersion in 4%paraformaldehyde for 24 hours.
The next day, discard the fixative and rinse the cell pellets with deionized water. Place one layer of gauze onto the tissue cassette and transfer the fixed spheroid pellets using a pipette. Cover the pellet by folding the gauze and close the cassette lid.
Next, dehydrate the spheroid pellets by immersion in 70%ethanol for five minutes at room temperature. Then, continue through a series of ethanol concentrations for five minutes each. Then immerse the pellets in 100%xylene for 15 minutes.
Immerse the spheroid pellets in paraffin at 56 degrees Celsius for embedding. Next, cut five micron-thick sections from the paraffin-embedded cell pellet using a rotary microtome. Float the sections in 50%ethanol, and then transfer them to a 50 degree Celsius water bath.
Mount the floating sections onto slides, and then proceed to safranin o and alcian blue for immuno-flourescent staining. To determine a suitable centrifugal gravity force, ASC's were spun at centrifugal gravity of zero, 300, 600, 1, 200, and 2, 400 times g for fifteen minutes. SOX9 mRNA expression was evaluated at 24 hours after CG stimulation.
A CG of 2, 400 times g significantly induced SOX9 mRNA expression. To evaluate the expression of chondrogenesis-related genes, total RNA was extracted from unstimulated ASC's, ASC's that had been centrifuged, or treated with TGF-beta-one. As seen here, CG stimulation induces collagen type-2 and aggrecan expression.
over-expression of collagen type-2 in ASC's treated with CG was confirmed by immunoflourescence using an antibody against collagen type-2 in an Alexa Fluor 594 conjugated secondary antibody. To determine the effect of CG on chondrogenesis-related ECM over-expression, ASC's were treated with TGF-beta-one as a positive control. The negative control was a pellet culture not subjected to CG loading, or TGF-beta-one treatment.
The over-expression of chondrogenesis-related ECM was evaluated in spheroid pellets consisting of centrifuged ASC's through the use of safranin o and alcian blue staining. Glycosaminoglycan, a major component of articular cartilage, was stained blue by alcian blue, or stained orange by safranin o. After 14 days of micromass culture, chondrocyte condensation was evaluated by the size of the aggregates.
The size of each aggregate, generated by CG, was around 0.5 millimeters. Once mastered, this technique can be done in 30 minutes, if it is performed properly. After watching this video, you should have a good understanding of how induce chondogenic differentiation of stem cells using centrifugal gravity force.
