Overview
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.
- 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.
- 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. - 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.
- 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. - 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.
- 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.
- 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.
- 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. - 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.- 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.
- Remove COCs from the dissolved capsules.
NOTE: This can be done with a cut pipette tip. - 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.
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Materials
Name | Company | Catalog Number | Comments |
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 |