연골 재생을위한 3D 자기 줄기 세포 집계 및 생물 반응기 성숙

The overall goal of this magnetic cell aggregation and dynamic maturation method is to improve the in vitro chondrogenesis of mesenchymal stromal cells or MSC's. Magnetic aggregation of stem cells instead of chondrogenesis as cell condensation is an early key step of the cartilage regeneration process. After the stem cells are confined into scaffolds the scaffolds can be moved into cellular bioreactor to assess dynamic maturation of the cells.

Important dynamics method can be applied to other bio-systems such as muscle, cardiac, or vascular repair. But as you will see, it is very well-suited for cartilage to regenerate. Visual demonstration of the magnetic cell seeding and the dynamic cell maturation using the actual bioreactor are critical because these two techniques are difficult to learn from text instruction only.

To construct the micromagnet device for aggregate formation first insert a 750 micrometer diameter magnetic tip into each three centimeter hole in a six millimeter thick aluminum plate and place the plate over a permanent neodymium magnet. To construct a device for scaffold seeding first cut hard polystyrene into 2.4 square centimeter squares and insert three millimeter diameter, six millimeter long magnets at an equal distance over a 1.6 square centimeter surface. Then place the device over a permanent neodymium magnet.

To label human MSC's, first culture the stem cells in complete MSC growth medium at 37 degrees Celsius at 5%carbon dioxide until they are 90%confluent. Aspirate and then rinse the cells with Serum three RPMI medium without glutamine, and add 10 milliliters of iron oxide nano particle solution per 150 square centimeter culture flask for a 30 minute incubation in the cell culture incubator. At the end of the incubation, rinse the cells for five minutes with Serum three RPMI medium without glutamine to internalize the nano particles still attached to the plasma membrane.

Then replace the RPMI in each flask with 25 milliliters of complete MSC growth medium and return the cells to the cell culture incubator overnight. The next morning, detach the magnetic cells with eight milliliters of 05%Trypsin-EDTA per flask. And collect the dissociated cells by centrifugation.

We suspend the pellets for counting and place a glass bottom 35 millimeter cell culture Petri dish on top of each magnetic device. To magnetically form the aggregates, add three milliliter of chondrogenic medium to each Petri dish and gently deposit no more than eight microliters of 2.5 times 10 to the 5th labeled cells per aggregate. It is critical that the cells are deposited neither too slowly nor too fast.

And neither too close nor too far from the magnetism. When all of the aggregates have been placed allow spheroids to form for 20-30 minutes without disturbance. Then transfer the aggregates to the cell culture incubator.

On the same day, form aggregates by centrifuging 2.5 times 10 to the 5th labeled stem cells in 15 milliliter conical tubes with 1.5 milliliters of chondrogenic medium. To magnetically seed the scaffolds place one dried pullulan/dextran polysaccharide porous scaffold per Petri dish and dilute 2 times 10 to the 6th labeled cells in 350 microliters of chondrogenic medium without TGFbeta3. Carefully pipette the cells onto the scaffolds and incubate the structures at 37 degrees Celsius to allow full cell penetration.

After five minutes gently add three milliliters of chondrogenic medium to the Petri dish and return the scaffolds to the cell culture incubator for four days to allow the cells to migrate through the scaffold pores. On the same day, also seed another set of scaffolds with 2 times 10 to the 6th labeled stem cells as demonstrated and incubate the cells without the magnet to obtain uniformly seeded scaffolds as positive controls. For chondrogenesis into scaffolds on day five remove the magnetic device from all the scaffolds and return the static scaffolds to the cell culture incubator until day 25 changing the medium twice a week.

For dynamic cell culture also on day five cut silicone tubing to the appropriate length according to the manufacturers instructions and pace the tubing, the 500 milliliter culture chamber the two way rotators and the cages into a sterile microbiological safety station. Following the manufacturers instructions connect the tubing to the two way rotators in the culture chamber. Then use a sterile spatula to carefully transfer two cellularized scaffolds into each cage.

When all of the scaffolds have been transferred insert the cages through the needles in the lid to keep them from moving during the rotation. And fill the culture chamber with chondrogenic medium. Use sterile antiseptic technique when preparing the chambers to avoid contamination of any of the tools or scaffolds.

Next insert the cages into the chamber and then close the chamber and turn on the peristaltic pump to fill the tubing with chondrogenic medium and eliminate air bubbles. Place and secure the filled chamber within the motor of the bioreactor and turn on the computer which controls the rotations of the arm and the chamber. Then apply a rotation speed of five rotations per minute to both the arm and the chamber, and adjust the peristaltic pump to a flow rate of 10 rotations per minute for continuous feeding of the cellularized scaffolds.

To generate a scaffold free 3D aggregate construct on day eight initiate the fusion by placing two aggregates in contact in chondrogenic medium to for eight dublets. Then on day 11 merge two dublets to form four quadruplets, and form the final structure on day 15 by fusing the four quadruplets. Tissues formed by magnetic fusion demonstrate a significant increase in collagen II expression compared to the pellets obtained by centrifugation.

With an increased trend of aggregant expression. For cellularized static scaffolds and increase in aggregant and collagen II expressions are observed when magnetic seeding is used, compared to the scaffold seeded without magnetic forces. Interestingly the expression of collagen II is much higher when magnetic seeding is combined with dynamic differentiation.

Histological analysis using toluidine blue reveals that the sequential magnetic fusion of 16 aggregates exhibits an abundance deposition of glucosamine glycans. Further in magnetically seeded scaffolds the glucosamine glycan content is higher when the scaffolds are differentiated in a bioreactor than when they are cultured statically. Once mastered the magnetic cell aggregation can be completed in two hours if performed properly.

When attempting this procedure, it is important to remember to resuspend the labeled cells in as small a volume as possible to facilitate the aggregate formation. Following the magnetic seeding of the scaffolds the dynamic maturation using the actual bioreactor can be performed to improve the expression new synthesized chondrocytes. Since it's development this technique paved the way for researchers in the field of regenerative medicine to explore the regeneration of articular cartilage in humans.

After watching this video you should have a good understanding of how to construct magnetic devices, label stem cells form stem cell aggregates, magnetically seed scaffolds and use an actual bioreactor. Don't forget that working with human cells and tissue can be extremely hazardous and that precautions should always be taken, such as wearing gloves, labcoats, and protective glasses.