3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads
Published: April 30, 2023
Abstract
Source: Gorczyca, G., et al. Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation. J. Vis. Exp (2020).
This video describes the protocols for encapsulating porcine cumulus-oocyte complexes (COCs) in fibrin-alginate beads to establish a 3D culture. This in-vitro maturation system maintains normal cell-to-cell communication, ensuring oocyte maturation and retaining viability.
Protocol
Encapsulation in fibrin-alginate hydrogel beads 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. 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. On the day of the procedure, thaw it slowly on ice, and right before the use, bring to room temperature. …
Declarações
The authors have nothing to disclose.
Materials
| General | |||
| Antibiotic Antimycotic (100x) 100ml | Thermo Fisher | 15240062 | 2.5% final concentration for Handling Medium. 1% in PBS (step 1.2) |
| DMEM/F12 (500ml) | Sigma-Aldrich | D8062 | Handling and Maturation Medium |
| DPBS (w/o Ca, Mg), 1x, 500ml | Thermo Fisher | 14190144 | |
| FCS (100 ml) | Thermo Fisher | 16140063 | 10% final concentration for both Handling Medium and Maturation Medium. (steps: 1.5. 2.6.) |
| PBS (1x, pH 7.4) 500ml | Thermo Fisher | 10010023 | |
| TBS Stock Solution (10x, pH 7.4) 500 ml | Cayman Chemicals | 600232 | 1x final concentration. Other brand can be use |
| Maturation Medium | |||
| hCG (1 VIAL of 10 000 U) | Sigma-Aldrich | CG10 | |
| PMSG | BioVendor | RP1782721000 | |
| Fibrin-alginate beads | |||
| Alginate Lyase | Sigma-Aldrich | A1603 | |
| Thrombin | Sigma-Aldrich | T9326-150UN | |
| Calcium Chloride | Sigma-Aldrich | C5670 | |
| Fibrinogen (250mg) | Sigma-Aldrich | F3879 | |
| Sodium Alginate | Sigma-Aldrich | W201502 | Use for alginate solution |
| Specific instruments, tools | |||
| 30 mm Pteri dish | TPP | 93040 | |
| 60 mm IVF Petri dish | Falcon | 353653 | |
| Ez-Grid Premium Cell Handling Pipettor | RI Life Sciences | 8-72-288 | |
| Ez-Tip | RI Life Sciences | 8-72-4155/20 | |
| Heating Table | SEMIC | Other brands can be used | |
| Incubator | Panasonic | MCO-170AIC-PE | Other brands can be used |
| Sterile petri dish (10 cm) | NEST Biotechnology | 704002 | |
| Sterile syringe filters with 0.2 µm | GOOGLAB SCIENTIFIC | GB-30-022PES | |
| Thermos | Quechua | 5602589 | Other brands can be used |
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3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads. J. Vis. Exp. (Pending Publication), e20843, doi: (2023).