3. Ideally, oocytes should be stored individually within straws and
the straws kept in at least two different locations so that should
an accident occur with one straw then all is not lost. Accurate
records should be kept of their location within the storage
vessel.
4. The straw can be filled using a small syringe and needle. Pushing the cotton plug along the straw will reduce the capacity of
the straw. The plug should be wetted as this will help prevent
liquid nitrogen entry. If the plug is wetted by insertion of a
sizeable column of the primary thawing/dilution solution
(in this case a 4 cm column of 1 M sucrose solution), then
expulsion of this solution along with the oocytes following
thawing will aid dilution of the CPA. An air gap (e.g., a
0.5 cm column) should be left between this section (1 M
sucrose solution) and the area to contain the oocytes—a 1 cm
column of CPA. Straws should be clearly labelled, with an
appropriate marker.
5. The straw contents can be expelled upon thawing by inserting a
1 mL syringe containing at least 0.5 mL of primary thawing/
dilution solution and expelling the contents of both the straw
and syringe into a dish.
6. Studies have been performed with oocytes that have been
denuded of cumulus cells (by means of treatment with hyaluronidase and/or gentle pipetting) prior to freezing or frozen
with the cumulus intact. No clear advantage either way is
evident. Human oocytes ought to be denudated within
37–40 h post-hCG administration for ovulation induction
and to be vitrified directly after denudation. Cryopreservation
of denuded oocytes is routine method; however it has been
shown that mouse oocytes with remaining intact cumulus cells
successfully survive VS in calcium-free medium with ethylene
glycol as CPA [72].
7. Faster cooling/warming rates can be achieved and the risk of
ice crystal formation reduced, by using straws that have been
pulled to reduce the wall thickness, or by use of such devices as
nylon loops or microscope grids. However, such devices are
even more susceptible to temperature change, and some of
these systems are “open,” i.e., the sample is in direct contact
with liquid nitrogen (see Note 12).
8. A piece of plastic that can be inserted into a straw and is
approximately twice the length of a straw is ideal. The plastic
is inserted into the end of the straw and then bent through 90
.
The straw can then be placed close to the nitrogen liquid
surface in the horizontal position, and the holder can be held
by hand in the cold nitrogen vapor at a safe distance from the
liquid nitrogen. A floating straws carrying device (CryoFloater,
Cryopreservation of Oocytes
449
the straws kept in at least two different locations so that should
an accident occur with one straw then all is not lost. Accurate
records should be kept of their location within the storage
vessel.
4. The straw can be filled using a small syringe and needle. Pushing the cotton plug along the straw will reduce the capacity of
the straw. The plug should be wetted as this will help prevent
liquid nitrogen entry. If the plug is wetted by insertion of a
sizeable column of the primary thawing/dilution solution
(in this case a 4 cm column of 1 M sucrose solution), then
expulsion of this solution along with the oocytes following
thawing will aid dilution of the CPA. An air gap (e.g., a
0.5 cm column) should be left between this section (1 M
sucrose solution) and the area to contain the oocytes—a 1 cm
column of CPA. Straws should be clearly labelled, with an
appropriate marker.
5. The straw contents can be expelled upon thawing by inserting a
1 mL syringe containing at least 0.5 mL of primary thawing/
dilution solution and expelling the contents of both the straw
and syringe into a dish.
6. Studies have been performed with oocytes that have been
denuded of cumulus cells (by means of treatment with hyaluronidase and/or gentle pipetting) prior to freezing or frozen
with the cumulus intact. No clear advantage either way is
evident. Human oocytes ought to be denudated within
37–40 h post-hCG administration for ovulation induction
and to be vitrified directly after denudation. Cryopreservation
of denuded oocytes is routine method; however it has been
shown that mouse oocytes with remaining intact cumulus cells
successfully survive VS in calcium-free medium with ethylene
glycol as CPA [72].
7. Faster cooling/warming rates can be achieved and the risk of
ice crystal formation reduced, by using straws that have been
pulled to reduce the wall thickness, or by use of such devices as
nylon loops or microscope grids. However, such devices are
even more susceptible to temperature change, and some of
these systems are “open,” i.e., the sample is in direct contact
with liquid nitrogen (see Note 12).
8. A piece of plastic that can be inserted into a straw and is
approximately twice the length of a straw is ideal. The plastic
is inserted into the end of the straw and then bent through 90
.
The straw can then be placed close to the nitrogen liquid
surface in the horizontal position, and the holder can be held
by hand in the cold nitrogen vapor at a safe distance from the
liquid nitrogen. A floating straws carrying device (CryoFloater,
Cryopreservation of Oocytes
449