Abstracts: CryoLetters 26 (2), 2005

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

CryoLetters 26 (2), 73-84 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


 Hans Ramløv1*, Arthur L. DeVries2 and Peter W. Wilson3

1 Department of Biology and Chemistry, Roskilde University, Roskilde, Denmark
2 Department of Animal Biology, University of Illinois, Urbana Champaign, IL, USA.
3 Department of Physiology, Otago Medical School, Dunedin, New Zealand.
* to whom correspondence should be addressed, E-mail:


This study investigates in detail the freezing events during cooling of solutions of various size classes of antifreeze glycoproteins.  Differential scanning calorimetry and nanolitre osmometry were used to observe ice growth at temperatures within the hysteresis gap between the melting point and non-equilibrium freezing point (hysteresis freezing point) of solutions of the various sized antifreeze glycoproteins (AFGPs). The ice growth within the hysteresis gap is presumably due to both the expression of primary or near primary prism planes and also some growth at the basal plane. The binding of the AFGPs to the ice causes a particular ice crystal morphology. With the smaller AFGPs (7,8) substantial microscopic ice growth was observed in the form of a hexagonal bipyramids within the hysteresis gap.

Keywords: antifreeze, thermal hysteresis, differential scanning calorimetry, nanolitre osmometer, ice.



CryoLetters 26 (2), 85-92 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Mina Ramezani1, Mojtaba Rezazadeh Valojerdi* 2,3, Kazem Parivar4

1Department of Biology, Ashtian Campus, Islamic Azad University, Tehran-Iran.
2Department of Anatomy, University of Medical Sciences, Tarbiat Modarres University, Tehran-Iran.
3Department of Embryology, Royan Institute, Tehran-Iran.
4Department of Biology, Science and Research Faculty, Islamic Azad University, Tehran-Iran.
Corresponding author: M.R. Valojerdi, Department of Embryology, Royan Institute, Tehran-Iran, P.O. Box: 19395-4644. E-mail:


The purpose of this study was to investigate the effect of three vitrification procedures [conventional straw (CS), open pulled straw (OPS) and closed pulled straw (CPS)] on the development of two-cell mouse embryos. Two-cell mouse embryos were randomly divided into vitrified and non-vitrified control groups. Embryos in the vitrified group were cryopreserved within a combination of 5.5 M ethylene glycol and 1M sucrose as cryoprotectants, loaded within three different straws (CS, OPS and CPS) and warmed in stepwise sucrose solutions. The survived embryos from each procedure were cultured in human tubal fluid (HTF). The non-vitrified control embryos were also cultured in the same manner. The rates of the development in all the groups were daily determined and statistically compared. On day 4 of the cultivation period, several expanded blastocysts from each group were randomly selected and stained either with propidium iodide (PI) and bisbenzimide or with terminal transferase- mediate DNA end labeling (TUNEL) Technique. The mean number of the inner cell mass (ICM), trophoectoderm (TE), necrotic and apoptotic cells were counted and statistically compared. The survival rate of embryos in CPS was significantly higher than that in OPS and CS. The rate of hatched blastocysts did not differ in the three vitrification procedures, but in comparison with that of the control, CS and OPS showed a significant reduction. The mean number of ICM and TE decreased in CS and OPS, whereas in CPS it was almost identical to that of the control. The incidence of apoptosis and necrosis appeared to be almost similar in all the groups. In conclusion, CPS seems to be an effective, easy and rapid method for the cryopreservation of two-cell mouse embryos.

Keywords: Vitrification, Closed pulled straw, Open pulled straw, Two-cell stage, Mouse embryos.



CryoLetters 26 (2), 93-102 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


S. Leunufna1 and E. R. J. Keller2*

1Pattimura University Ambon, Indonesia. Present address: Genebank Dept., Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
2Genebank Dept., Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany. E-mail:


Cryopreservation of yams (Dioscorea spp.) is important for the preservation of valuable genotypes for food, medicine and breeding purposes. This study on four species of yams was conducted to evaluate the influence of cold acclimation in an alternating 5°C / 28°C, 12 h thermo-photo-period and of two sucrose concentrations in the preculture medium using a modified droplet method. Acclimation of a 3-week period provided the best preconditioning treatment averaged over four genotypes. Effect of sucrose concentration in the preculture medium depended on the genotype (significant genotype x sucrose interaction; P = 0.036). High survival (67-70%) with 30% to 50% shoot recovery was obtained for D. bulbifera, D. polystachya and D. cayenensis, compared to 20% survival without shoot recovery for D. alata.

Keywords: Dioscorea, modified-droplet method, buds, cold acclimation, sucrose



CryoLetters 26 (2), 103-112 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jung-Bong Kim1, Haeng-Hoon Kim1*, Hyung-Jin Baek1, Eun-Gi Cho1, Yong-Hwan Kim1 and Florent Engelmann2,3

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea (email for H.H. Kim:
2Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies. (present address). 
3International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


Changes in moisture content (MC), sucrose and glycerol concentration in garlic shoot tips were monitored during loading and unloading with PVS3 solution. Upon PVS3 treatment, shoot tip MC decreased rapidly and sucrose and glycerol concentrations increased rapidly during the first 30 min. Sucrose and glycerol concentrations increased more slowly thereafter. Shoot tip MC in after PVS3 treatment was affected by their size, but not by sucrose concentration of the preculture medium. As the size of shoot tips increased, so their MC increased after PVS3 treatment.  However, sucrose and glycerol concentrations decreased after PVS3 incubation, and concentrations in dehydrated shoot tips were much lower than those measured in non-air dried controls. During unloading with 1.2 M sucrose medium, shoot tip MC increased rapidly during the first 10 min, whereas glycerol concentration decreased steadily over 90 min. Loading and unloading of PVS3 solution in garlic shoot tips follows the principle of solute bulk flow.

Keywords: Allium sativum L., HPLC, sucrose, glycerol, PVS3, vitrification



CryoLetters 26 (2), 113-120 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Aili Zhang1*, Shuxia Cheng2, Dayong Gao3 and Lisa X. Xu1,4

1School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, PR China.
2Department of Thermal Science and Energy Engineering, University of Science & Technology of China.
3Department of Mechanical Engineering, University of Washington, Seattle, USA.  4School of Mechanical Engineering, Purdue University, USA.


Two methods used in artery deep cryopreservation, “Cryopreservation in Medium” and “Cryopreservation in Air”, were studied. For the former method, samples were frozen together with a certain amount of cryoprotectants (CPA) in the cryovial or cryobag, while for the other method the arteries were first exposed to CPA and then frozen without the CPA medium surrounding in the cryovial. Study of the cryopreserved arteries using these two methods found that “cryopreservation in air” could substantially reduce the fracture rate of the arteries (12). To explain the difference theoretically, a two-compartment model is presented to study the thermal stresses generated during the freezing and thawing processes. The properties were measured as inputs to the model. Numerical results showed that the thermal stresses occurring in the “cryopreservation in air” process were much smaller than in the other method. The maximum thermal stress during cryopreservation occurs in the thawing process. The theoretical results could well explain published experimental results. 

Keywords: artery, thermal stress, cryopreservation in air, cryopreservation in medium



CryoLetters 26 (2), 121-130 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Survival of FOUR ACCESSIONS OF Anigozanthos manglesii (Haemodoraceae) seeds FOLLOWING exposURE to liquid nitrogen

D. J. Merritt1*, D.H. Touchell2, T. Senaratna1,3, K.W. Dixon1,3 and C. W. Walters4

1Kings Park and Botanic Garden, West Perth, WA 6005, Australia.
2School of Forestry and Wood Products, Michigan Technological University, Houghton, MI 49931-1295, USA.
3Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA, 6009, Australia.
4USDA-ARS National Centre for Genetic Resources Preservation, 1111 South Mason St, Fort Collins, CO 80521, USA.


This study investigated the survival of seeds from the prominent endemic Western Australian species Anigozanthos manglesii following exposure to liquid nitrogen (cryostorage). Seeds from four different accessions (collected in 1987, 1990, 1993 and 1998) adjusted to different water contents were tested for survival following cryostorage. Water content was a significant determining factor with survival of cryostored seeds declining rapidly at water contents above c. 18%. These water contents were deemed as critical water contents and were supported by DSC scans showing high endothermic peaks indicating ice crystallisation. In some instances, survival of cryostored seeds also declined at low water contents. Seeds from 1990 had a lower than expected survival compared to the other accessions. This may have resulted from the higher lipid content of seeds from this accession, or the reduced germination and vigour of these seeds prior to cryostorage.

Keywords: Anigozanthos manglesii, conservation, cryostorage, seed, water content.



CryoLetters 26 (2), 131-136 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


A.Neronov1*, J. Mazgalova2, M.Cholakova1, M.Dimitrova1, I. Deligiozova2, S.Kovatcheva2 and E.Nikolova1

1Institute Experimental Morphology and Anthropology, Bulgarian Academy of Sciences, acad. G.Bonchev str., bl. 25, 1113 Sofia, Bulgaria. 
2Eye, Tissue and Cell Bank,University hospital “Queen Giovanna”, 8 Bialo more str., 1527 Sofia, Bulgaria.


The aim of the present study was to elaborate an optimal method for cryopreservation of human donor cornea for transplantation and to follow the morphological changes in the structure of the endothelial cell layer using scanning electron microscopy (SEM). Sixteen groups, with four donor cornea each, were cryopreserved at cooling rates of 1°/min and 5°/min. Four cryoprotectants (glycerol, dimethyl sulfoxide, 1,2-propanediol, polyethylene glycol-400) in two concentrations – 5% and 10 % v/v, were prepared on the bases of medium Optisol GS supplied with 20 % v/v human serum albumin. Four additional human cornea were used as controls. Endothelial cell recovery of the cornea after thawing and 24 hours culture, was calculated as a percent of the preserved recovered cells. Sufficient recovery of the endothelial cell layer, making the cornea suitable for transplantation was obtained using the cryoprotectants dimethyl sulfoxide and especially polyethylene glycol-400.

Keywords: human cornea, cryopreservation, SEM observation.

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