Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.
This protocol for encapsulation of porcine oocytes makes it possible to maintain spherical organization of COCs. This prevents their flattening and consequent disruption of gap junctions between the oocyte and surrounding follicular cells. The main advantages of the two described protocols are that are user-friendly and allow for effective control of both the oocyte and chemo cell survival.
Both of the culture systems are valuable tools for basic research in reproductive biology, and may have clinical relevance for improved infertility treatment. They can also be applied for developing novel biotechnology methods and for livestock improvement. After rinsing the ovaries, transfer them to a beaker filled with HM medium and store them in an incubator at 38 degrees Celsius during all subsequent manipulations.
Aspirate the follicular fluid from large porcine follicles and centrifuge it at 100 times G for 10 minutes at room temperature. Then, filter the supernatant using a sterile syringe attached to a 0.2-micrometer membrane pore filter, and snap freeze it at negative 80 degrees Celsius. To isolate the COCs from medium-sized follicles, transfer two to three ovaries to a sterile 10 centimeter Petri dish filled with HM.Gently cut the surface of the protruding ovarian follicles with a sterile surgical number 15 blade, which will cause the follicular fluid and the COCs to flow out into the Petri dish.
Aspirate the follicular contents with a 28 gauge needle attached to a disposable syringe, and transfer it into another Petri dish. To prepare the IVF Petri dishes, add one milliliter of HM to the central wells, and place three to four drops of HM in the outer rings. Then use a polycarbonate micropipette to move the undamaged COCs to drops of HM in the outer rings, and briefly rinse them three to four times.
When finished, individually transfer them into the central well. Store the IVF plates in the incubator. 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 one to one ratio for a final volume of two milliliters and gently vortex the mixture. Repair 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 eight to 10 drops arranged in two rows. Use a micropipette to transfer three to five 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 at 38 degrees Celsius incubator for five to seven minutes.
After incubation, transfer each FA capsule into a well of a 96-well plate containing 100 microliters of maturation medium. Every two days, replace half of the medium with fresh pre-equilibrated maturation medium. Image COC is 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 international units per milliliter algenate 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. Make a bed of five grams of FEP powder in a 30 millimeter Petri dish. Distribute a single droplet of maturation medium containing three to five 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 three to four milliliters of sterile water to the outer rings to create a humidity chamber. Pick up the formed LM with a 1000 microliter pipette and place it into the central well.
Incubate the marbles for four days at 38 degrees Celsius in a five percent 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 bio-reactor to a drop of fresh maturation medium in the Petri dish.
After three to four 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. Both in vitro maturation systems produced COCs with granulosis cells that were tightly adhered to each other and intact layers of cumulus cells.
The COC viability analysis confirmed that optimal growth conditions were achieved in both encapsulation systems. In both groups, only high viability oocytes sites were observed. The oocytes were imaged with transmission electron microscopy.
The mitochondria were evenly distributed and had a shell-like shape. Only a few of them were elongated and their clustering was sporadically observed. Endoplasmic reticulum was either associated with mitochondria or free in the oocyte cytoplasm.
Liquid droplets appeared as small, dark, round structures, and Golgi apparatus were emerged with dilated cisternae. It is important to be precise and careful when transferring FA capsules from incubation chamber into culture plates and when picking up and transferring format LM into the IVF Petri dish.
