In vitro growth of mouse preantral follicles under the simulated microgravity. The whole procedure can be divided into four parts. Part one, mechanically isolate follicles from the ovaries using needles under stereomicroscope.
Part two, encapsulate single follicle by pipetting it into the middle of each alginate bead. Part three, transfer the alginate beads containing the single follicles into the culture media droplet in the rotating culture system which generates the simulated microgravity. Part four, pick up alginate beads and decapsulate the follicles from the alginate beads by incubating in medium supplemented with Alginate lyase for 30 minutes at 37 degrees Centigrade.
Follicle isolation and encapsulation. Collect the ovaries in handling medium and then further place the ovaries in a centered well plate. Under a stereomicroscope, the follicles are isolated from the ovaries by two needles.
Take care not to impair the vasomembrane. Prepare a dish with droplets of medium and cover it with the oil. Transfer the follicles to the prepared dish.
Wash the follicles in the new medium and then transfer them into the droplets of medium. One droplet of medium contains a single follicle. Prepare a centered culture dish add the encapsulation solution into the outer circle.
Prepare a vial of the zero point eight percent of alginate solution. Add zero point five microliter of alginate solution into the center of the dish. Add droplets of the alginate solution into the encapsulation solution in the outer circle of the dish.
Transfer the follicle into the alginate solution in the center of the dish. Rinse the follicles with the alginate solution. Transfer the follicles into the middle of each alginate bead through the soft surface of the solution.
Each alginate bead contains a single follicle. Rinse the alginate beads with the encapsulation solution to solidify the beads. Transfer the alginate beads to a new dish with handling medium.
In vitro culture of encapsulated follicles under simulated microgravity treatment. For control group, add 150 milliliter culture medium to a new dish and cover it with oil. Transfer three alginate beads into the medium droplet.
Incubate the dish in a dioxide incubator for culture. For simulated microgravity treatment, prepare a dish of oil, remove the port cap, add the oil into the vessel through the open port, until the vessel is nearly full. Add one 50 milliter culture medium into the oil.
Transfer three alginate beads into the medium droplet. Replace the open port cap. The air bubbles in the vessel must be removed.
Remove the syringe port cap. Place two syringe with oil on the syringe port. Maneuver the air bubbles underneath the syringe port.
Pull the bubbles into the syringe and inject the same volume of oil out of the other syringe. Make sure there is no air bubbles in the vessel. Close the valves, remove the syringes and replace the syringe port cap.
We have already prepared four vessels with alginate beads. Now it is time to fix the vessels into the rotating base. Adjust the rotation rate until the medium droplet's in a state of balance.
Transfer both the vessels and the base into a dioxide incubator for culture. Changing culture medium. Remove the open port cap, collect the alginate beads and the oil in a new dish.
Find and transfer the alginate beads into a dish with the handling medium. Reestablish the vessel culture system as before. Follicle retrieval.
Remove the open port cap, collect the alginate beads and the oil in a new dish. Pick up the alginate beads and decapsulated follicles from the alginate beads by incubating in handling medium, supplemented with an alginate lyase for 30 minutes at 37 degrees centigrade.Results. Cell viability assay showed that the single follicles cultured at normal gravity condition has more live cells and less dead cells than the follicles cultured at simulated microgravity conditions.
The follicle size increased significantly under both gravity conditions.Conclusion. Our method provides a model to study the mechanisms involved in the in vitro developmental processes of oocyte and granulosa cells under simulated microgravity condition.
