Liquid Marble Microbioreactor System: A 3D Culture Protocol for Encapsulation of Porcine Oocytes

Make a bed of 5 grams of FEP powder in a 30-millimeter Petri dish. Distribute a single droplet of maturation medium, containing 3 to 5 COCs onto the FEP powder. Rotate the plate gently in a circular motion to ensure that the particles completely cover the surface of the liquid drop and form liquid marbles or LMs.

Prepare several 60-millimeter IVF Petri dishes and add 3 to 4 milliliters of sterile water to the outer rings to create a humidity chamber. Pick up the formed LM with a 1,000 microliter pipette and place it into the central well. Incubate the marbles for four days at 38 degrees Celsius in a 5% carbon dioxide incubator.

To change the medium, apply 30 microliters of maturation medium onto each LM, which will cause it to spread. When the marble contents dissolve, transfer the COCs released from the bioreactor to a drop of fresh maturation medium in the Petri dish. After 3 to 4 washes in maturation medium, transfer the COCs along with 30 microliters of fresh medium, onto the FEP powder bed.

Gently rotate the plate in a circular motion to ensure that the powder particles completely cover the surface of the liquid drop and form a new LM. Then, transfer the LM to the IVF Petri dish.