This procedure uses a three-dimensional cell culture system to mimic in vivo cell kinetics. Begin by incubating cells with collagen ENC coated beads. Then load the cells and beads into a rotating cell culture system and propagate 3D cell aggregates in a low shear, low turbulence environment.
Finally, harvest the cells for use in downstream applications compared to gene expression and cellular differentiation of cells grown in a typical monolayer. The resulting 3D cultures demonstrate cell kinetics and behavior that closely resemble that scene. In vivo.
We have used the RCCS to engineer biologically meaningful 3D models of various human epithelial tissues. Dr.Jessica Warner, a former grad student in my collaborator, Dr.Sydney Morris's lab, will now demonstrate this procedure with EVT cells In a 50 milliliter conical tube autoclave 250 milligrams of micro carriers cyto dex three beads in 12 milliliters. DPBS allow the prepared beads to swell and cool to room temperature.
Then under sterile conditions, bring the total volume to 12.5 milliliters. Using DPBS now harvest an 80%confluent culture of extra villus trophoblast cells. Using trypsin digestion gently mix the prepared cyto dex three beads and using a wide tip 10 milliliter serological pipette transfer 2.5 milliliters to a 15 milliliter conical tube.
After the cyto dex three beads have settled to the bottom of the tube. Pipette off the top layer of DPBS without disturbing the cyto Dex three beads. Mix the EVT cells with the cyto Dex three beads.
Incubate at room temperature for 30 minutes with periodic mixing, followed by 30 minutes in cell culture incubator with occasional mixing. Place a 10 milliliter RCCS into a sterile six well culture plate and remove the large stopper. Now add warmed GTSF two media to bring the total volume of the cell bead mixture to 10 milliliters and load into the RCCS through the large port.
Replace the large port stopper and close small ports. Next position, empty three milliliter syringes onto the two small ports. Add media to each syringe.
Slowly open the two valves and replace the syringe pistons. Gently add media from one syringe until all air bubbles are expelled from the inside chamber. Pick up the RCCS and gently tap the side while rotating it to check for bubbles.
Remove remaining bubbles by rotating the RCCS until the bubbles are under the small port. Then gently push down on the syringe on the opposite side to force the bubbles into the port and out of the chamber. Continue to close one side port gently push down on the other syringe piston to introduce a small amount of positive pressure into the vessel and close the second valve while applying pressure.
Finally load the RCCS onto the rotor. Start the rotation at 19 RPM in a cell culture incubator for optimal culture conditions. Replenish the media every other day for the first three days and daily thereafter.
First turn off the rotor and remove the RCCS. Pull the pistons up to create some suction. Remove the syringes from each small port and place the RCCS on an angle and allow the beads to settle opposite of the large port.
Now, open one of the small valves to release two thirds of the media from the RCCS into a waste container without disturbing the beads. Close the small valve, then open the large port. Add media into the RCCS and replace the stopper, expel all air bubbles as shown earlier, and position the culture onto the rotator in the incubator.
Once the aggregates start growing, visibly increase rotation speed to ensure cells remain in suspension. To harvest the cells. Release the RCCS from the rotor.
Discard the syringes from each small port, and place the RCCS on an angle to direct beads to opposite the large port. After the beads have all settled, open one of the small valves and drain a third of the media into a waste container. Close the small valve and open the large port.
Gently swirl the vessel to disperse the aggregates back into solution and transfer the cells into a sterile 50 milliliter conical tube. After allowing the collected aggregates to settle in the conical tube, use the supernatant to thoroughly wash the culture vessel again to maximize recovery of aggregates. Using a wide bore pipette tip immediately aliquot aggregates for downstream functional assays.
The S-G-H-P-L four trophoblast cell line is one example of extra villus trophoblasts like cells. In a typical aggregate grown in an RCCS on cyto X three, many beads are completely covered with propagating cells. Note the projections extending away from the main cluster and attaching to neighboring clusters.
Once removed from the RCCS and plated on an extracellular matrix, the EVT like 3D grown cells aggressively invade and or migrate. Indeed, R-T-P-C-R data confirms the increased expression of MMPs seen in 3D aggregates as opposed to traditional cell culture monolayers. Interestingly, genes not associated with invasion are also upregulated in the RCCS.
After watching this video, you should have a clear understanding of how to use the RCCS with your cell line primary cells or even stem cells to obtain three dimensional cell cultures that more closely mimic cell kinetics in vivo.
