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

Prepare a bed of fluorinated ethylene propylene, or FEP, powder in a culture dish. Add a drop of maturation medium containing cumulus-oocyte complexes, or COCs, onto the FEP powder. Gently roll the drop over the powder to form a liquid marble, or LM. The FEP powder creates a hydrophobic porous shell, allowing gas exchange between the interior liquid and the surrounding environment.

Now, collect the LM unit and transfer it into the central well of an IVF Petri dish. Add sterile water to the outer ring to keep the COCs hydrated. Then, incubate the LM under physiological conditions to allow oocyte maturation. 

Next, remove the spent medium and add a fresh maturation medium. Direct liquid contact disrupts the hydrophobic FEP shell and breaks the LM to release the COCs. Transfer the released COCs to a new dish containing a fresh drop of maturation medium and wash them to remove FEP particles.

After washing, transfer the COCs and the fresh medium onto the FEP powder bed. Roll the drop to coat it with powder. Finally, transfer the LM to the IVF Petri dish. FEP powder-coated LMs maintain the 3D structure of the oocytes and enhance their viability.