Nidacon, Sweden), that provides a constant distance between
the straws and the nitrogen surface, thereby allowing freezing
in the cold nitrogen vapor, can be also the option.
9. Addition of the high concentrations of CPA used in VS solutions directly to the PBS medium will result in reduction of the
salt concentrations within that medium. Using a concentrated
PBS medium allows the CPA to be added prior to the addition
of water to give the required volume of single strength PBS
medium with appropriate isotonic salt balance.
10. The VS solution described here could be replaced with other
combinations of CPA, for example, ethylene glycol together
with sucrose, or ethylene glycol plus propanediol plus sucrose
often in combination with a macromolecule such as Ficoll or
trehalose, which has been used successfully for the preservation
of bovine and human oocytes, or in combination with newly
applied cryoprotective agent carboxylated ε-poly-L-lysine
resulted in production of live offspring in mice [73]. Exposure
to this CPA mixture can be performed at 37
C thereby avoiding any cooling-related damage.
11. In order to adhere to the timing of each step of the procedure,
oocytes should be processed in small batches.
12. It should be noted that straws allow rapid heat transfer and
hence are susceptible to temperature change during handling,
for example, on transfer from the cooling machine to liquid
nitrogen for storage and during handling for identification
purposes prior to thawing. Straws can be stored in the liquid
or vapor phase of liquid nitrogen. If stored within the liquid
phase, care should be taken to prevent leakage of liquid nitrogen into the straw as this will expand on warming and may
cause the straw to explode or crack. Also, the liquid nitrogen
may be contaminated with viruses, which have been shown to
survive such temperatures. Straws can also be placed within a
second vessel, for example, a second larger straw (straw in straw
method), to reduce liquid nitrogen entry. One end of the larger
straw should be pre-sealed and plunged into liquid nitrogen,
then VS straw containing oocytes placed inside of it and
another end of the covering straw accurately sealed avoiding
warming of the sample. However, debate continues about the
impact on cooling and warming rates which enclosure in a
second straw produce, and whether this has an impact on
oocyte survival [6]. If storing in the vapor phase is chosen,
the level of liquid nitrogen in the storage dewar should be
carefully monitored to ensure stability of storage temperature.
Alarms and automatic filling systems are available, but it is
recommended that the level of liquid nitrogen still be checked
manually at regular intervals.
450
Victoria Keros and Barry J. Fuller
the straws and the nitrogen surface, thereby allowing freezing
in the cold nitrogen vapor, can be also the option.
9. Addition of the high concentrations of CPA used in VS solutions directly to the PBS medium will result in reduction of the
salt concentrations within that medium. Using a concentrated
PBS medium allows the CPA to be added prior to the addition
of water to give the required volume of single strength PBS
medium with appropriate isotonic salt balance.
10. The VS solution described here could be replaced with other
combinations of CPA, for example, ethylene glycol together
with sucrose, or ethylene glycol plus propanediol plus sucrose
often in combination with a macromolecule such as Ficoll or
trehalose, which has been used successfully for the preservation
of bovine and human oocytes, or in combination with newly
applied cryoprotective agent carboxylated ε-poly-L-lysine
resulted in production of live offspring in mice [73]. Exposure
to this CPA mixture can be performed at 37
C thereby avoiding any cooling-related damage.
11. In order to adhere to the timing of each step of the procedure,
oocytes should be processed in small batches.
12. It should be noted that straws allow rapid heat transfer and
hence are susceptible to temperature change during handling,
for example, on transfer from the cooling machine to liquid
nitrogen for storage and during handling for identification
purposes prior to thawing. Straws can be stored in the liquid
or vapor phase of liquid nitrogen. If stored within the liquid
phase, care should be taken to prevent leakage of liquid nitrogen into the straw as this will expand on warming and may
cause the straw to explode or crack. Also, the liquid nitrogen
may be contaminated with viruses, which have been shown to
survive such temperatures. Straws can also be placed within a
second vessel, for example, a second larger straw (straw in straw
method), to reduce liquid nitrogen entry. One end of the larger
straw should be pre-sealed and plunged into liquid nitrogen,
then VS straw containing oocytes placed inside of it and
another end of the covering straw accurately sealed avoiding
warming of the sample. However, debate continues about the
impact on cooling and warming rates which enclosure in a
second straw produce, and whether this has an impact on
oocyte survival [6]. If storing in the vapor phase is chosen,
the level of liquid nitrogen in the storage dewar should be
carefully monitored to ensure stability of storage temperature.
Alarms and automatic filling systems are available, but it is
recommended that the level of liquid nitrogen still be checked
manually at regular intervals.
450
Victoria Keros and Barry J. Fuller