Abstracts: CryoLetters 27 (5), 2006

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

CryoLetters 27 (5), 269-282 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

comparison of three techniques for cryopreservation and reestablishment of long-term Gentiana tibetica suspension culture

Anna Mikuła

Botanical Garden Center for Biological Diversity Conservation, Polish Academy of Sciences, ul. Prawdziwka 2, 02-973 Warsaw, Poland


Cryogenic storage of cell suspensions allows long-term maintenance of cultures. The main purpose of the study was to develop a successful cryogenic protocol for 10-year-old embryogenic cell suspensions of G. tibetica. We examined three techniques of freezing: (I) controlled-rate cooling with various cryoprotectants (0.1-0.5 M DMSO, 0.5-1.0 M sucrose, 0.5-1.0 M glycerol, 0.25-1 M proline) or preculture with 0.4 M sorbitol and cryoprotectants (0.065-0.1 M DMSO, 0.2-0.8 M proline), (II) vitrification (PVS2) and (III) encapsulation. Cell viability was assessed by the TTC test and biomass increase. After controlled-rate cooling the majority of cells were lethally damaged, with only 3% viability observed. Vitrification and encapsulation approaches were more effective, assuring high levels of post-thaw viability ca. 85% and 70%, respectively. The encapsulation procedure gave faster recovery of the culture suspension than did vitrification, and ensured culture homogeneity and embryogenic competence.

Keywords: Gentiana tibetica, cell suspension, cryopreservation, programmed freezing, vitrification, encapsulation



CryoLetters 27 (5), 283-290 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Аlexander К. Gulevsky, Liana I. Relina* and Yelena А. Grishchenkova

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, Pereyaslavskaya Street, 23, 61015 Kharkov, Ukraine.


Data relating to the peculiarities of antioxidant enzyme activities, glutathione (GSH) level and lipid peroxidation (LP) intermediates specific to at each ontogenic stage of Tenebrio molitor are presented. Metamorphosis is accompanied by a shift of prooxidant-antioxidant balance towards oxidative processes, since pupae have the highest levels of lipid peroxidation intermediates. Cold acclimation (4C) can promote oxidative stress at the cold sensitive developmental stage imagoes, which enhance their levels of diene conjugates and ketodienes after a 2-week cold acclimation. This enhancement is accompanied by an increase in catalase (CAT) activity. GSH levels undergo no changes in imagoes after cold acclimation. Neither larval nor pupal T. molitor show significant changes in LP product contents after cold acclimation. Chilling results in a significant increase in CAT activities in pupae, but not in larvae. GSH levels are reduced both in larvae and pupae after cold exposure. However, cold acclimation does not affect superoxide dismutase (SOD) activity in any of the developmental stages of T. molitor.

Keywords: lipid peroxidation, antioxidant system, ontogenesis, cold impact, Tenebrio molitor



CryoLetters 27 (5), 291-294(2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


K.K. Newsham*, N.R. Maslen and S.J. McInnes

British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 0ET, U.K.
* Email:


A sample of the liverwort Cephaloziella varians was collected on 1 January 1999 at Rothera Point on the Wright Peninsula, Adelaide Island, western Antarctic Peninsula and was partially dried and then frozen at -80C. The sample was rapidly defrosted to c. 10C after six years and two months of storage at this temperature. Nematodes, tardigrades and a bdelloid rotifer present in the sample were found to have survived. Of the 159 nematodes recovered from the sample, 49 (31%) were alive: of the tardigrades and rotifers, two of 15 (13%) and one of 48 (2%) had survived, respectively. A Chi-square test showed that there was a significant association between nematode taxon and survival: a greater proportion of Coomansus gerlachei individuals were alive than of Rhyssocolpus paradoxus. A Chi-square test also showed that there was a significant association between phylum and survival: a significantly greater proportion of nematodes or tardigrades were alive than of bdelloid rotifers. We conclude that Antarctic soil metazoans are capable of surviving long-term exposure to low sub-zero temperatures and that there may be taxon-specific effects of freezing on survival.

Keywords: bdelloid rotifers, Coomansus gerlachei, freezing, nematodes, Rhyssocolpus paradoxus, tardigrades



CryoLetters 27 (5), 295-303(2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

The effect of cold-acclimation on the water relations and freezing tolerance of Hordeum vulgare l.

S. Burchett1*, S.Niven2 and M P. Fuller3

1University of Plymouth, School of Biological Science, B439, Portland Square, Plymouth PL4 8AA
2Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
3University of Plymouth, Graduate School, Room 4, Mary Newman Building, Plymouth PL4 8AA


During a 5C and a 5/-1C cold acclimation (CA) regime there was a significant decline in the water potential of winter barley, and a concurrent decline in tissue water content of the 5/-1C CA plants. Results of carbohydrate analysis illustrated a significant (P<0.001) accumulation of sucrose, fructose and glucose in the 5/-1C CA plants, which was inversely correlated to water potential. Using an infrared imaging radiometer during a convection frost test the water release time (WRT) of 5/-1C CA was demonstrated to be significantly (P<0.001) longer than that observed in non-cold acclimated plants. This observation is consistent with visual analysis of exotherm curves where the rate of cellular water release to extracellular ice is reduced in the 5/-1C CA plants, compared to the non-cold acclimated plants. These biochemical and physiological changes were correlated to increased plant health following a non-lethal freezing test to -5oC, where non-cold acclimated plants produced 2.3 0.33 tillers and 5C and 5/-1C CA plants produced 2.4 0.33 and 4.7 0.67 tillers, respectively.  Results from this study imply that cold acclimation leads to changes in the physical state of water that result in a less osmotically responsive cellular environment and subsequently significantly less damage to meristematic tissue.

Keywords: Water potential, water content, carbohydrate analysis, infrared imaging radiometer, water release time.



CryoLetters 27 (5), 305-310 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jun Kasuga, Kaoru Mizuno, Natsuko Miyaji, Keita Arakawa and Seizo Fujikawa*

Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan. *Corresponding author


In order to find the possible role of intracellular contents in facilitating the supercooling capability of xylem parenchyma cells, changes in the temperature of supercooling levels were compared before and after the release of intracellular substances from beech xylem parenchyma cells by DTA. Various methods were employed to release intracellular substances from xylem parenchyma cells and all resulted in a reduction of supercooling ability. It was concluded that the reduction of supercooling ability primarily resulted from changes of intracellular conditions, including the release of intracellular contents or their mixing with extracellular solutions, rather than due to changes of cell wall structures. It is therefore suggested that any unidentified intracellular contents may function to facilitate supercooling capability in xylem parenchyma cells.

Keywords: Deep supercooling, xylem parenchyma cells, cold acclimation, intracellular freezing



CryoLetters 27 (5), 311-318 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Thiagarajan Hemalatha1, Vairakkannu Vaijayanthi Mala1, Bhakthavatsalam Murali Manohar2, Mohammed Nayeem3, Samu Subramaniam4 and Rengarajulu Puvanakrishnan1*.

1Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai 600 020, India,
2Department of Pathology, Madras Veterinary College, Vepery, Chennai, India,
3Alved Pharma & Foods Pvt. Ltd., Chennai, India, and
4Department of Biochemistry, Apollo Hospitals, Greams Lane, Chennai, India.
*Tel: +91 044 24430273 Fax: +91 44 491 1589 Email:


 Cryoinfarction in rats was carried out by placing the cooled probe directly on the midway of left anterior descending coronary artery. This caused damage to the blood vessel, hindering blood flow to the distal part of the left ventricle. Gross pathology showed around 26 % infarction at 24 h. Histopathology revealed death of cardiomyocytes with blood vessel congestion at the end of 24 h, inflammatory infiltrate at 48 h, fibrotic scar by 96 h and collagen deposition by 192 h. Acute myocardial infarction biomarkers such as Cardiac Troponin T, Creatine Kinase MB and NT pro BNP were shown to be elevated by 4 h. ECG showed an ST segment elevation by 96 h.  This cryoinfarction model was suitable to study changes taking place during acute myocardial infarction in humans.

Keywords: acute myocardial infarction, coronary artery ligation, cTnT, CK-MB, NT pro BNP.



CryoLetters 27 (5), 319-328 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

zebrafish embryos (Danio rerio) using microinjection

Julia Kopeika, Tiantian Zhang, David Rawson*

Institute of Research in the Applied Natural Sciences, University of Bedfordshire, The Spires, 2 Adelaide Street, Luton, LU1 5DU, United Kingdom


Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P<0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20C to 0C at 2C/min, from 0C to 7.5C /min at 1C/min, seeded at -7.5C and held for 10 min, before cooling at 0.3C/min to - 20C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy.  The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.

Keywords: zebrafish (Danio rerio), embryos, microinjection, intracellular crystallization



CryoLetters 27 (5), 329-332 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


J. Cardona-Costa *, J. Roig, M. Perez-Camps and F. Garca-Ximnez

Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Camino de Vera 14, 46071 Valencia, Spain.


No data on vitrification of tissue samples are available in fishes.  Three vitrification solutions were compared: V1: 20% ethylene glycol and 20% dimethyl sulphoxide; V2: 25% propylene glycol and 20% dimethyl sulphoxide, and; V3: 20% propylene glycol and 13% methanol, all three prepared in Hanks buffered salt solution plus 20% FBS, following the same one step vitrification procedure developed in mammals.  Caudal fin tissue pieces were vitrified into 0.25 ml plastic straws in 30s and stored in liquid nitrogen for 3 days minimum, warmed (10s in nitrogen vapour and 5s in a 25C water bath) and cultured (L-15 plus 20% FBS at 28.5C). At the third day of culture, both attachment and outgrowing rates were recorded. V3 led to the worst results (8% of attachment rate). V1 and V2 allow higher attachment rates (V1: 63% vs V2: 50%. P < 0.05) but not significantly different outgrowing rates (83%-94%).  Vitrification of caudal fin pieces is advantageous in fish biodiversity conservation, particularly in the wild, due to the simplicity of procedure and equipment.

Keywords: Cryopreservation, vitrification, cryoprotectants, zebrafish, tissue culture, biodiversity.

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