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