X-Message-Number: 26665
Date: Tue, 19 Jul 2005 19:14:06 -0700 (PDT)
From: Doug Skrecky <>
Subject: Supercool X100 abstracts

Cryobiology. 2005 Jun;50(3):325-31.
Anomalous high activity of a subfraction of polyvinyl alcohol ice
blocker.
Wowk B
21st Century Medicine, Inc., 10844 Edison Court, Rancho Cucamonga,
CA 91730, USA.
  Low molecular weight copolymers of polyvinyl alcohol (PVA) are
known to be potent inhibitors of ice formation in solutions used
for cryopreservation by vitrification, even at concentrations as
low as one part per million. Concentrated aqueous solutions of
these polymers tend to become turbid after preparation. Condensed
particles causing turbidity were isolated from a commercially
available PVA-based ice blocker (X-1000) and found to consist of a
polymer subfraction that is especially effective at ice blocking.
Fifty seven percentage (w/w) of ethylene glycol (EG) in distilled
water and 0.025% of the condensate polymer showed similar stability
against devitrification as 57% EG+0.1% ordinary X-1000. At higher
concentrations, 56.9% EG+0.1% condensate polymer was as effective
as 56% EG+1% ordinary X-1000. All solutions containing ice blocker
showed much less devitrification during warming than a 57% EG
control solution. The condensate polymer was found to be strongly
self-associating and less water soluble than ordinary X-1000. The
mean molecular weight of the condensate polymer was approximately
1400 compared to 2100 for ordinary X-1000. Proton NMR revealed no
large chemical differences. Subtle differences in composition or
stereochemistry, perhaps in local regions of molecules, must be
responsible for the dramatic differences in physical behavior and
ice blocking effectiveness of the condensate polymer.

Plant Cell Rep. 2005 Apr 28; [Epub ahead of print]
An efficient cryopreservation procedure for potato (Solanum
tuberosum L.) utilizing the new ice blocking agent, Supercool X1000.
  Cryopreservation has been recognized as a practical and efficient
tool for long-term storage of vegetatively propagated plants. This
study was conducted to investigate effects of modified vitrification
techniques on cryopreservation of potato. In vitro plants of potato
cultivars 'Superior' and 'Atlantic' were cold acclimated, and
axillary buds were precultured, osmoprotected, exposed to PVS-2
solution, plunged into liquid nitrogen, thawed, and finally planted
in the regeneration medium. In the modified vitrification technique
an ice-blocking agent, Supercool X1000, was added with PVS-2 solution.
Cold acclimation affected survival of cryopreserved shoot tips, and
the highest survival (46.7%) was obtained after 3 weeks of
acclimation at 10 degrees C. Shoot tips exposed to 2M glycerol plus
0.6M sucrose for 40 min gave 51.5% and 11.7% survival in 'Atlantic'
and 'Superior' at 10 degrees C, respectively. Cold acclimated and
osmoprotected shoot tips were dehydrated with PVS-2 containing
different concentrations of Supercool X1000 prior to a plunge into
liquid nitrogen. Treatments with 0.1% and 1% of Supercool X1000
significantly improved survival by 55% in 'Superior' and 71.3% in
'Atlantic', respectively. After cryopreservation, vitrified shoot
tips resumed growth within a week in a medium (1 mg l(-1) GA(3),
0.5 mg l(-1) zeatin, and 0.1 mg l(-1) IAA) with a low level of
Pluronic F-68 (0.005%) and survival was 33.7% higher in 'Atlantic'
and 14.7% higher in 'Superior' than the control (without Pluronic F-68).

Cryobiology. 2000 May;40(3):228-36
Vitrification enhancement by synthetic ice blocking agents.
Wowk B
21st Century Medicine, Inc., Rancho Cucamonga, CA 91730, USA.
  Small concentrations of the synthetic polymer polyvinyl alcohol
(PVA) were found to inhibit formation of ice in water/cryoprotectant
solutions. Ice inhibition improved with decreasing molecular weight.
A PVA copolymer of molecular weight 2 kDa consisting of 20% vinyl
acetate was found to be particularly effective. PVA copolymer
concentrations of 0.001, 0.01, 0.1, and 1% w/w decreased the
concentration of glycerol required to vitrify in a 10-ml volume
by 1, 3, 4, and 5% w/w, respectively. Dimethyl sulfoxide
concentrations required for vitrification were also reduced by
1, 2, 2, and 3% w/w, respectively. Crystallization of ice on
borosilicate glass in contact with cryoprotectant solutions was
inhibited by only 1 ppm of PVA copolymer. Devitrification of
ethylene glycol solutions was also strongly inhibited by PVA
copolymer. Visual observation and differential scanning calorimeter
data suggest that PVA blocks ice primarily by inhibition of
heterogeneous nucleation. PVA thus appears to preferentially bind
and inactivate heterogeneous nucleators and/or nascent ice crystals
in a manner similar to that of natural antifreeze proteins found in
cold-hardy fish and insects. Synthetic PVA-derived ice blocking
agents can be produced much less expensively than antifreeze
proteins, offering new opportunities for improving cryopreservation
by vitrification.

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