3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads

Prepare thrombin and fibrinogen solutions as described in the text manuscript. On the day of the procedure, slowly thaw the fibrinogen solution on ice, and bring it to room temperature right before use. Mix 0.5% alginate solution and the fibrinogen solution at a 1:1 ratio for a final volume of 2 milliliters, and gently vortex the mixture.

Prepare incubation chambers by applying thin strips of paraffin film to glass microscope slides. Pipette 7.5-microliter drops of the FA mixture onto the paraffin-film-coated glass slide with separating spacers, placing 8 to 10 drops arranged in two rows.

Use a micropipette to transfer three to 5 COCs to the center of the FA drop, along with a minimum volume of maturation medium. Add 7.5 microliters of thrombin solution on top of each FA drop to cover it. There's no need to mix them because the gel forms almost instantaneously.

Cover the incubation chamber with a previously prepared glass slide. Then, turn the chamber upside down, and place it in a 100-millimeter Petri dish lined with moist filter paper. Put the Petri dish in the 5% Carbon Dioxide (38 C) incubator for five to seven minutes.

After incubation, transfer each FA capsule into a well of a 96-welled plate containing 100 microliters of maturation medium. Every two days, replace half of the medium with fresh, pre-equilibrated maturation medium. Image COCs using an inverted light microscope at 10x magnification.

After culturing the COCs for four days, remove the maturation medium from the wells, and add 100 microliters of 10 IU/mL alginate lysate in DMEM. Leave the culture plate in the incubator for 25 to 30 minutes. Remove the COCs from the dissolved capsules. Wash them in DMEM several times. Then, transfer them to the inner ring of an IVF dish containing PBS.