PVS2 are two cryoprotectant solutions that are used for plant cell
cryopreservation for three decades. In their study, four solutions
were rapidly cooled to liquid nitrogen temperatures, and observations were made subsequently upon warming. As illustrated in
Fig. 8, rapid cooling was sufficiently fast to achieve vitrification,
but the warming rate at À10
C per min was not rapid enough to
avoid devitrification of the PVS1 solution and VSL solution upon
warming above À80
C. PVS2 and VSL+ are more stable during
warming and have much smaller devitrification exotherms (arrows)
due to the increase in the concentration of glycerol, DMSO,
and/or sucrose.
DSC studies using a series of cooling and warming rates permit
one to determine the critical cooling and warming rate to achieve
vitrification and to avoid devitrification. Suzuki et al. [7] further
studied the effect of cooling rates. Cooling rates between
À100
C/min and À10
C/min did not affect the warming thermograms of vitrified solutions, demonstrating that two new solutions (VSL and VSL+) could be vitrified even at moderate cooling
rates. VSL is less viscous than PVS2 because of its lower glycerol
concentration and higher ethylene glycol concentration, permitting
faster penetration and/or faster dehydration of tissues with a
potentially wide applicability to cryopreservation of plant-cultured
materials and genetic resources. For verification, VSL and VSL+
solution were tested for cryopreservation of gentian axillary buds.
VSL exhibited 78% survival as determined by the regrowth capacity,
which was comparable to PVS2 and PVS1. VSL had a wider optimal
incubation time (20–45 min) than PVS2 and was more suitable for
cryopreserving gentian buds.
4.4 Ice Damage Upon
Cryopreservation
DSC can be used to investigate the mechanism of actions for the
effect of desiccation, conditioning, and other treatments on the
survival of cells and tissues after cryopreservation. Figure 9 is an
example that tests the encapsulation of osmotically preconditioned
tissues (shoot tips) in alginate and then desiccation to a tolerable
lower water content in order to increase the glass transition temperature and to minimize ice damage during cooling [8]. The
investigators characterized thermal profiles of silica gel desiccated
encapsulated shoot tips of Ribes nigrum upon cooling and warming
(see Note 8). Five hours of desiccation reduced the water content of
shoot tip samples from ~3.8 to ~0.4 g per gram dry mass and
increased the glass transition temperature to ca. –80
C, and a
small amount of ice formation was observed from the warming
curve. It achieved >60% post-cryopreservation viability. Seven
hours of desiccation reduced the water content further to ~0.3 g
per gram dry mass, increased the glass transition temperature to
ca. –55
C, completely eliminated ice formation, and achieved
ice-free cryopreservation.
DSC Analysis of Thermophysical Properties
297
cryopreservation for three decades. In their study, four solutions
were rapidly cooled to liquid nitrogen temperatures, and observations were made subsequently upon warming. As illustrated in
Fig. 8, rapid cooling was sufficiently fast to achieve vitrification,
but the warming rate at À10
C per min was not rapid enough to
avoid devitrification of the PVS1 solution and VSL solution upon
warming above À80
C. PVS2 and VSL+ are more stable during
warming and have much smaller devitrification exotherms (arrows)
due to the increase in the concentration of glycerol, DMSO,
and/or sucrose.
DSC studies using a series of cooling and warming rates permit
one to determine the critical cooling and warming rate to achieve
vitrification and to avoid devitrification. Suzuki et al. [7] further
studied the effect of cooling rates. Cooling rates between
À100
C/min and À10
C/min did not affect the warming thermograms of vitrified solutions, demonstrating that two new solutions (VSL and VSL+) could be vitrified even at moderate cooling
rates. VSL is less viscous than PVS2 because of its lower glycerol
concentration and higher ethylene glycol concentration, permitting
faster penetration and/or faster dehydration of tissues with a
potentially wide applicability to cryopreservation of plant-cultured
materials and genetic resources. For verification, VSL and VSL+
solution were tested for cryopreservation of gentian axillary buds.
VSL exhibited 78% survival as determined by the regrowth capacity,
which was comparable to PVS2 and PVS1. VSL had a wider optimal
incubation time (20–45 min) than PVS2 and was more suitable for
cryopreserving gentian buds.
4.4 Ice Damage Upon
Cryopreservation
DSC can be used to investigate the mechanism of actions for the
effect of desiccation, conditioning, and other treatments on the
survival of cells and tissues after cryopreservation. Figure 9 is an
example that tests the encapsulation of osmotically preconditioned
tissues (shoot tips) in alginate and then desiccation to a tolerable
lower water content in order to increase the glass transition temperature and to minimize ice damage during cooling [8]. The
investigators characterized thermal profiles of silica gel desiccated
encapsulated shoot tips of Ribes nigrum upon cooling and warming
(see Note 8). Five hours of desiccation reduced the water content of
shoot tip samples from ~3.8 to ~0.4 g per gram dry mass and
increased the glass transition temperature to ca. –80
C, and a
small amount of ice formation was observed from the warming
curve. It achieved >60% post-cryopreservation viability. Seven
hours of desiccation reduced the water content further to ~0.3 g
per gram dry mass, increased the glass transition temperature to
ca. –55
C, completely eliminated ice formation, and achieved
ice-free cryopreservation.
DSC Analysis of Thermophysical Properties
297