REMOVAL OF CUMULUS CELLS USING HYALURONIDASE
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PREPARATION OF HYALURONIDASE STOCK SOLUTION
Materials
Sterile
saline
Sterile dolphin-nose microcentrifuge tubes
Sterile
5-10 ml beaker
Preparation
- Prepare stock solution
of 10,000-units/ml hyaluronidase in sterile
saline in a sterile beaker.
- Sterilize the stock
solution by filtering through a 0.2 mm syringe filter into
another sterile beaker or sterile tube.
- Prepare aliquots by
adding 100 ml of stock solution
into sterile microcentrifuge tubes.
- Freeze at -20°C
We use this concentration of hyaluronidase and it works fine. However, several different concentrations are used in the literature and it might be worthwhile to test several concentrations empirically to decide which concentration works best for each individual laboratory.
PROCEDURE
FOR CUMULUS CELL REMOVAL
- Place 1 aliquot (100 ml) of hyaluronidase stock in the incubator/oven (38.5°C) at least 2 hours
prior to using. (Fig. A)
- At time of cumulus
cells removal, add 900 ml HEPES-TALP (this will
make a working concentration of 1000 units/ml hyaluronidase). (Fig B)
- Add cumulus-oocytes complexes (COC) to the dolphin-nose microcentrifuge tube. (Fig C) We add up to
300 COC/tube. Thaw more aliquots of
hyaluronidase if more than 300 COC are to be
processed.
- After all COC have been
placed in the tube, place the tube on a slide warmer or incubator for 1-2
minutes to let all the COC settle at the bottom of the tube.
- Carefully aspirate the
excess hyaluronidase/HEPES-TALP solution down to
the top of the nose without disturbing the COC. (Fig. D)
- To make sure none have
been lost, check the aspirate for COC and recover any found.
- Vortex for 5 minutes
(refer to IVP protocol)
A B C D
COC
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