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. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=26665