This article describes a method for oocyte maturation using liquid marbles formed from fluorinated ethylene propylene (FEP) powder. The technique enhances the viability of cumulus-oocyte complexes (COCs) during in vitro fertilization (IVF) by maintaining their 3D structure.
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.
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.
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