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. …

Açıklamalar

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

Etiketler

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Bu Makaleden Alıntı Yapın
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).

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