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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Encapsulation in super-hydrophobic fluorinated ethylene propylene (FEP) microbioreactors.

NOTE: Make sure that all in vitro maturation (IVM) procedures are carried out on a thermostatically controlled table, and cumulus-oocyte complexes (COCs) are maintained at 38 °C throughout their handling.

1. Prepare a 30 mm Petri dish containing 5 g of FEP powder bed-average particle size of 1 μm
   NOTE: It is important to use fresh FEP powder. The re-used FEP tends to aggregate and form clumps.
2. Distribute a single droplet of maturation medium (MM) (~30 μL in volume) containing 3-5 COCs onto the bed of the FEP powder. Rotate the plate gently in a circular motion to be sure that these particles completely covered the surface of the liquid drop and liquid marbles formed (LM).
3. Pick up formed LM using a pipette with a 1,000 μL tip.
   NOTE: Cut the pipette tip at the edge to accommodate it to the diameter of the LM (4-5 mm). The approximate diameter of such a prepared tip should be slightly bigger than that of the LM.
4. Prepare several 60 mm in vitro fertilization (IVF) Petri dishes. Add 3-4 mL of sterile water to the outer rings to prepare the humidity chamber. Next, place one LM into the central well.
   NOTE: This procedure requires good manual skills and precision – if the marble is placed carelessly or falls even from a low height, it will be destroyed.
5. Incubate marbles for 4 days at 38 °C in a 5% CO₂ incubator.
   1. Change the medium daily following the procedure: apply 30 μL of MM on each LM, which will cause their spreading because direct liquid contact disrupts the hydrophobicity of the coated FEP powders. When the marble content dissolves, transfer COCs released from the bioreactor to a drop of fresh MM in the Petri dish.
      NOTE: To precisely transfer released COCs, use a polycarbonate micropipette.
   2. After several washes in MM (3-4 times) to remove FEP particles, transfer them with 30 μL of fresh MM 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.
   3. Pick up formed LM using a pipette with a 1,000 μL tip.
   4. Add 3-4 mL of sterile water to the outer rings to prepare the humidity chamber. Next, place one LM into the central well of the IVF Petri dish.
      NOTE: The transparent coating of FEP powder allows to monitor LM content using a light microscope.