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

Encapsulation in fibrin-alginate hydrogel beads

1. Prepare 1 mL of 50 IU/mL thrombin in Tris-buffered saline (TBS; pH 7.4) with 100 mM CaCl₂ in a 2.0 mL sterile microcentrifuge tube.
2. Prepare fibrinogen stock solution (50 mg/mL in TBS) and keep it frozen.
   NOTE: To avoid clumping when dissolving fibrinogen in TBS, heat TBS to 37 °C.
   1. On the day of the procedure, thaw it slowly on ice, and right before the use, bring to room temperature.
3. Just before use mix at a 1:1 ratio 0.5% alginate solution and 50 mg/mL fibrinogen solution to get finally 2 mL (FA). In a sterile microcentrifuge tube gently vortex the mixture. Avoid making bubbles.
4. To prepare "incubation chambers", apply thin strips of paraffin films to the glass microscope slides.
   NOTE: Wipe the slides with 70 % EtOH before use. One incubation chamber is formed with two slides. To separate them, place 3 mm spacers on one of them.
5. Pipette 7.5 μL drops of the FA mixture onto this paraffin film-coated glass slide, which also has separating spacers. On one slide, place 8-10 drops, arranged in two rows.
6. Transfer, using a micropipette, 3-5 cumulus oocyte complex (COCs) along with a minimum volume (up to 5 µL) of maturation medium (MM), and place them precisely in the center of the FA drop. This procedure requires good manual skills and precision.  
   NOTE: The maturation medium (MM) consists of DMEM/F12, supplemented with 10 IU/mL PMSG, 10 IU/mL hCG, 10% FBS and 50% porcine follicular fluid (FF) collected in step 1.4.
7. Add 7.5 μL of thrombin/Ca²⁺ solution on each FA drop, just to cover them. There is no need to mix them because the gel forms almost instantaneously.
8. Cover the incubation chamber by applying the second, previously prepared glass slide to the FA capsules. With a very careful but firm movement, turn the chamber upside down and place it in a 100 mm Petri dish lined with moist filter paper to avoid drying.
9. Then, transfer the Petri dish to the 5% CO₂ incubator (38 °C) for about 5-7 min. After this, FA capsules will become cloudy because of simultaneous gelation of fibrin and alginate.
10. Transfer FA capsules into 96 well plates (one capsule per well) containing 100 μL MM per well.
    NOTE: To not damage the formed FA capsules, perform this step carefully with the use of surgical forceps.
11. Every 2 days replace half of the MM (50 μL) with fresh, pre-equilibrated one. Image COCs using an inverted light microscope (at 10x magnification).
    NOTE: Culture conditions for COCs FA culture: 38 °C, under a 5% CO₂ atmosphere and relative humidity 95%. After 4 days of IVM, the hydrogels appear almost clear due to progressive degradation of the fibrin component. The remaining alginate should be degraded enzymatically – by alginate-lyase, a plant-derived enzyme that specifically degrades alginate and does not affect animal cells.
    1. Remove MM from wells containing the capsules and add 100 μL of 10 IU/mL alginate lyase in DMEM. Leave the culture plate in the incubator for 25-30 min.
    2. Remove COCs from the dissolved capsules.
       NOTE: This can be done with a cut pipette tip.
    3. After several washes in a fresh DMEM, transfer COCs to the inner ring of an IVF dish (5-10 COCs per dish) containing PBS. Now, use them for further morphological or biochemical analysis.