Abstracts: CryoLetters 27 (2), 2006

CryoLetters is a bimonthly, international journal for low temperature science and technology

CryoLetters 27 (2), 65-72 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Kaori Touno1*, Kayo Yoshimatsu2 and Koichiro Shimomura1, 3

1Plant Regulation Research Center, Toyo University, 1-1-1, Izumino, Itakura-machi, oura-gun, Gunma, 374-0193, Japan
2Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, 1 Hachimandai, Tsukuba, Ibaraki, 305-0843, Japan
3 Faculty of Life Sciences, Toyo University, 1-1-1, Izumino, Itakura-machi, oura-gun, Gunma, 374-0193, Japan
*Corresponding author; e-mail


Atropa belladonna hairy roots (clone M8) were successfully cryopreserved by using the vitrification method.  A. belladonna hairy root tips were precultured on a half strength of Murashige and Skoog (MS) solid medium with 0.1 mg/L 2,4-D or without phytohormone for 1 day, and then dehydrated with PVS2 solution for 15 minutes prior to immersion into liquid nitrogen for 1 day, 1 week, 1 month and 3 months. Hairy root tips kept in liquid nitrogen were rapidly thawed at 36 °C in a water bath. The root tips were recultured on 1/2 MS medium. The hairy root tips, precultured with 2,4-D before cryopreservation, showed a higher survival rate than those precultured without phytohormone.  The hairy root tips, precultured with 2,4-D, showed an average survival rate of 83%.  There was no significant difference in the viability of the hairy roots cryopreserved for different periods. The regrowth of cryopreserved hairy roots was similar to that of untreated hairy roots and tropane alkaloid productivity became stable after 4th subculture.  PCR analysis of hairy roots demonstrated the conservation of the T-DNA in cryopreserved hairy roots. These results indicate that cryopreservation by vitrification method is useful to preserve A.belladonna hairy root clone M8.

Keywords: cryopreservation, Atropa belladonna, hairy root, tropane alkaloids, vitrification method, T-DNA



CryoLetters 27 (2), 73-86 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Charles J. Hunt1,4, David E. Pegg2 and Susan E. Armitage3

1 UK Stem Cell Bank, National Institute for Biological Standards and Control Blanche Lane, Potters Bar, Herts EN6 3QG.
2 Medical Cryobiology Unit, Department of Biology, University of York, York YO1 5DD, UK.
3 NBS London Cord Blood Bank, Deansbrook Road, Edgware, Middlesex HA8 9BD, UK.
4 The work described in this paper was undertaken at NBS Tissue Services with a grant supplied by the Joely Bear Appeal. To whom correspondence should be addressed at: UK Stem Cell Bank, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire, EN6 3QG.


Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50%. In the context of umbilical cord blood such losses are unacceptable. Whilst an empirical approach can provide an acceptable level of recovery, the cryopreservation process can only be optimised by adopting a methodological approach. This paper provides an overview of just such an approach as illustrated by a study on CD34 cells from umbilical cord blood. It involves firstly the determination of membrane permeability parameters that can then be used to model “safe” addition and elution protocols for the chosen cryoprotectant, in this case dimethyl sulphoxide. This in turn permits cryoprotectant toxicity to be evaluated free from the confounding effect of osmotic damage caused by inappropriate addition and elution protocols. Finally, non-toxic concentrations of cryoprotectant may be investigated in a cooling rate study to provide an optimal cryopreservation protocol. Using the model, the effect on CD34 cells of current addition and elution protocols was also examined.

Keywords: Cord blood; haematopoietic stem cells; CD34; cryopreservation; cryoprotectant toxicity; freezing injury



CryoLetters 27 (2), 87-98 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Changes in Intracellular Potassium and Sodium Content of 2-Cell Mouse Embryos Induced by Exposition to Vitrification Concentrations of Ethylene Glycol§

Alexander G. Pogorelov1*, Igor I. Katkov2, Evgenia I. Smolyaninova3, Dmitri V.Goldshtein1

1Institute of Theoretical and Experimental Biophysics, RAS, 142290 Puschino, Moscow Region, Russia.
2UCSD Cancer Center, San Diego, California 92122, USA.
Institute for Problems of Cryobiology and Cryomedicine, NASU, 61015 Kharkov, Ukraine.
§Results of this work were in part presented at the International Conference “Preservation of Genetic Resources”, St. Petersburg, Russia, October  19-22, 2004 , and at the 42nd Annual Meeting of the Society for Cryobiology, Minneapolis, MN, USA, July 24-27.


Intracellular concentration of potassium and sodium in two-cell mouse embryos in G1/S phase after exposition to vitrification solutions containing ethylene glycol (EG) and sucrose or after incubation in Dulbecco`s solution were measured by electron probe microanalysis (EPMA). The embryos at room temperature were treated in 10% EG for 10 min, transferred into mixture of EG and 1.0 M sucrose in ratio of 3:7 (v/v) for 3 min, then to 0.5 M sucrose for 10 min followed by washing the cells with Dulbecco`s solution for 10 min prior to analysis. The cytoplasmic concentration of potassium and sodium in controlled untreated with EG embryos were in a range of 116-130 mM of potassium and 120 mM of sodium, with good concordance in two identical experiments. After exposition that mimicked vitrification protocols, the intracellular potassium dropped almost two-three-fold (47 + 3 mM in one experiment and to 70 mM in the second experiment. The intracellular sodium concentration also decreased two-fold in range 60-70 mM after treatment with EG. Possible mechanisms of changes in the intracellular elemental concentrations including the high intracellular sodium observed in intact embryos are discussed..

Keywords: intracellular potassium; intracellular sodium; early mouse embryo; vitrification; ethylene glycol; electron probe microanalysis (EPMA).



CryoLetters 27 (2), 99-106 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Implementation of garlic cryopreservation techniques in the National Plant Germplasm System

Dave Ellis1*, Dianne Skogerboe1, Christina Andre1, Barbara Hellier2
and Gayle Volk1

1National Center for Genetic Resources Preservation, USDA-ARS, 1111 South Mason Street, Fort Collins, CO 80521,
2Western Regional Plant Introduction Station, 59 Johnson Hall, Pullman, WA  99164


The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15% v/v DMSO, 15% v/v ethylene glycol, 30% v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50% v/w sucrose, 50% v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40% of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants.  Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.

Keywords: Allium sativum, garlic, genebanking, cryopreservation, PVS2, PVS3



CryoLetters 27 (2), 107-114 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Effect of tetrasodium tripolyphosphate on the freeze-concentrated glass-LIKE transition temperature of sugar aqueous solutions

Kiyoshi Kawai 1* and Toru Suzuki 2

1 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
2 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan.
* To whom correspondence should be addressed (e-mail:


The freeze-concentrated glass-like transition temperatures (Tg2s), so-called “ante-melting temperature” or “ice-melting temperature”, of tripolyphosphate-sugar aqueous solutions prepared with various sugars (ribose, sorbitol, glucose, maltose, sucrose, and trehalose) were investigated by using differential scanning calorimetry to evaluate the effect of tetrasodium tripolyphosphate on the Tg2 of sugar aqueous solutions. The Tg2s of tripolyphosphate-sugar aqueous solutions were higher than those of tripolyphosphate or sugar aqueous solutions and converged in a narrow temperature range of 238 to 243 K. Furthermore, a study of the Tg2s of tripolyphosphate-glucose aqueous solutions adjusted to various ratios indicated that the Tg2 increment depended on the ratio and that another glass-like transition appeared at a temperature below the Tg2 by increasing the ratio of tripolyphosphate. The drastic increase in the Tg2s of sugars with the addition of tripolyphosphate will be useful for improving the cryostabilization of biomaterials.

Keywords: tripolyphosphate, sugar aqueous solution, freeze-concentrated glass-like transition, cryostabilization, DSC



CryoLetters 27 (2), 115-126 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Gang Zhao1*, Xue-fei Bai2, Da-Wei Luo3 and Da-Yong Gao4

1 Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei 230027, P.R. China;
2 Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, P.R. China;
3 Department of Chemical Engineering, Texas A & M University, College Station, TX 77840;
4 Department of Mechanical Engineering, University of Washington, Seattle, WA 98195.


A multidimensional, finite element analysis (FEA) for the freezing, holding, rewarming and heating processes of biological tissues during the cryosurgery process of the new Combined Cryosurgery/Hyperthermia System is presented to theoretically test its validity. The tissues are treated as nonideal materials freezing over a temperature range, and the thermophysical properties of which are temperature dependent. The enthalpy method is applied to solve the highly nonlinear problem. It was found that when the same boundary condition and the same target tissue presented, the novel Cryosurgery/Hyperthermia System could supply the target tissue an approximative cooling rate, a much lower minimal temperature, a much greater warming rate, and a much greater thermal gradients compared with that of the simplified Endocare system. The numerical simulation indicates that the novel combined cryosurgery and hyperthermia system can provide an excellent curative effect in the corresponding cryotherapy. And the most attractive feature of this FEA framework is that it can be easily mastered by the surgeon without in-depth theory of heat transfer to analyze the cryosurgery process beforehand due to the friendly GUI (graphical user interface) of Ansys software.

Keywords: Cryosurgery/Hyperthermia, FEA, Ansys



CryoLetters 27 (2), 127-132 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Barry J Fuller1*, Colin Shurey2, Nick Lane1 Alex Petrenko3 and Colin Green2.

1University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, 
2Surgical Research Division, Northwick Park Institute for Medical Research, Harrow HA1 3UJ, UK
3Department of Biochemistry, Institute for Problems of Cryobiology and Cryomedicine, Ukraine Academy of Sciences, Kharkiv 61015, Ukraine.


Renal preservation at for 24 hours at hypothermia was studied in a rabbit model after flush cooling with sucrose-based solution (SBS), compared with a standard preservation solution (in this case, Marshall’s Hypertonic Citrate solution - HCA). Polyethylene glycol supplementation to SBS (SBS-PEG) was also investigated. Renal function was measured by plasma creatinine assays during 1 months post transplantation, and pathology of the explanted kidneys was undertaken. Results showed that survival at 28 days was similar in all groups, (HCA – 3/6; SBS – 2/5; SBS-PEG – 3/5), and there were no differences in recovery of  plasma creatinine values. Histopathological evaluation of the grafts indicated that SBS preservation resulted in more severe damage after transplantation (P<0.05 in both cortico-medullary region and medulla compared to HCA), whilst addition of PEG reduced the damage score to that seen with HCA. SBS can be used as a simple, inexpensive preservation solution for kidney cold storage provided that PEG is used as an additional colloid.

Keywords: Kidney; renal cold preservation; sucrose-based solution; polyethylene glycol; kidney transplantation, rabbit

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