This article describes the establishment of a three-dimensional culture of porcine cumulus-oocyte complexes (COCs) using alginate and fibrinogen biopolymers. The method facilitates communication between oocytes and cumulus cells, promoting cytoplasmic differentiation and maturation of oocytes.
The cumulus-oocyte complex, or COC, consists of a centrally located oocyte surrounded by closely arranged clusters of cumulus cells. To establish a 3D culture of porcine COCs, begin with a mix of alginate and fibrinogen biopolymers in the desired ratio.
Pipette drops of this mix onto a wax-coated glass slide. Seed this drop with pre-prepared COCs present in a suitable maturation media. This step ensures an adequate supply of nutrients from the media to the COCs.
Cover the polymer drop with the crosslinking solution containing thrombin and calcium ions. Place the spacers in the corners of the COC-containing slide and cover it with a fresh wax-coated slide to avoid shearing the polymer beads. Invert this slide assembly onto a petri dish and incubate.
During incubation, thrombin cleaves the fibrinogen into shorter fibrin fragments, which intercalate between the alginate polymer strands with the help of calcium ions. This results in the formation of a three-dimensional, interpenetrating polymer network, or IPN, encapsulating the COCs. Incubate the COC-polymer beads in the maturation media for a prolonged duration.
The IPN maintains oocyte-to-cumulus cells communication. This facilitates cytoplasmic differentiation involving organelle redistribution in the oocyte, which generates mature oocyte within the COC. Treat COC-polymer beads with the alginate-lyase enzyme to dissolve the IPN capsule, which then releases mature COCs. Transfer mature COCs to a fresh dish for further analysis.
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.
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