Abstracts: CryoLetters 26 (1), 2005

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

CryoLetters 26 (1), 1-6 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Heejaung Kim1,3, Kazuhito Itamoto2, Satoshi Une3, Munekazu Nakaichi3, Yasuho Taura3, and Sajio Sumida4

1 The United Graduate School of Veterinary Sciences, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.  
2 Department of Veterinary Internal Medicine and
3 Departments of Veterinary Surgery, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, and
4 Sumida Laboratory of Cryomedicine and Blood Transfusion, Kenketsu-Kyokyu Bldg. 1F, Tateishi 5-11-16, Katsushika, Tokyo, 124-0012, Japan.


Phosphoenolpyruvate (PEP) is a phosphorylated glycolytic intermediate that can penetrate the RBC membrane and be metabolized to 2,3-DPG and ATP. In this study, we evaluated the effects of PEP treatment on canine red blood cells (RBCs) cryopreserved with 12.5% (w/v) HES. RBCs were incubated for 30, 60, and 90 min at 37°C with PEP solution containing 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, 1 mM adenine and 50 mM PEP (340 m osm/kg), pH 6.0 and then cryopreserved in liquid nitrogen with 12.5% (w/v) HES for 2 weeks. 2,3-DPG and saline stabilities of the PEP treated groups were increased and osmotic fragility indices were significantly decreased compared to the untreated control group. There were no differences in 2,3-DPG levels within the PEP treated groups with different PEP incubation times. These results suggest that PEP treatment may be beneficial for the cryopreservation of canine RBCs with HES.

Keywords: canine red blood cells, cryopreservation, hydroxyethyl starch, phosphoenolpyruvate



CryoLetters 26 (1), 7-16 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Peitao Wang1, 2, Zhiquan Shu1, Liqun He1, Xiangdong Cui3, Yuzhen Wang2*, and Dayong Gao1,3*

1 Department of Thermal Science and Energy Engineering (TSEE), University of Science and Technology of China (USTC), Hefei, 230027, China. 
2 School of Life Science, University of Science and Technology of China (USTC), Hefei, 230027, China.
3 Department of Mechanical Engineering and Center for Biomedical Engineering, University of Kentucky, Lexington, Kentucky, 40506-0108, USA. 


HELAs (Hela cells, passed cells of human cervical carcinoma) were heat or cold treated (named heat or cold shock) and then resumed normal culture for 2, 4 or 8 hours respectively. The expressions of heat shock protein 70 (HSP70) and 90 (HSP90) of the HELAs were measured by Northern and Western blotting. HELAs after 4-hour culture were exposed to or cryopreserved with different concentration of dimethyl sulfoxide (Me2SO, 2.5%, 5%, 10%, 15% and 20% respectively, V/V). Meanwhile, the HELAs after different culture time (2, 4 and 6 hours of culture) were cryopreserved with 5% Me2SO. After exposure or cryopreservation, the number of live HELAs was counted and the survival rate was calculated. The results showed that heat shock increased the expression of HSP70 and HSP90 of HELAs, while cold shock decreased the expression of the two proteins. When the concentrations of Me2SO were 10%, 15% and 20%, the survival rates of HELAs after exposure to Me2SO or cryopreservation were much lower than those when the concentrations were small. The survival rates of the heat shocked HELAs were significantly higher than those of the cold shocked and control HELAs. After cryopreservation with 5% Me2SO, the survival rate of heat shocked HELAs group with 2 hours culture time was the lowest among all the groups of HELAs with different cultural time. From the results of this study, we conclude that the expressions of HSP70 and HSP90 in HELAs increased significantly after heat shock, while cold shock decreased the expressions of these two proteins. The over-expressions of HSPs in the heat shocked HELAs could protect the cells from both injury caused by potential toxicity of high concentrations of Me2SO and cryoinjury caused by the freeze-thawing/ cryopreservation procedure.

Keywords: HELAs, Heat shock, Heat shock protein (HSP), Cryopreservation, Me2SO



CryoLetters 26 (1), 17-24 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


I. Clavero-Ramírez1, J. Gálvez-Farfán1, J.M. López-Aranda1, and M.E. González-Benito2*

1Centro de Investigación y Formación Agraria de Málaga, IFAPA, Consejería de Innovación, Ciencia y Empresas, Junta de Andalucía, 20140-Churriana, Málaga, Spain
2Departamento de Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain (


This paper presents results from a study to develop cryopreservation procedures for apices of several strawberry genotypes. Five Fragaria x ananassa Duch. cultivars and two wild species (F. chiloensis and F. virginiana) have been screened using the encapsulation-dehydration method and/or a protocol which compromises vitrification and encapsulation-dehydration. Apices were encapsulated in an alginate gel, precultured on media containing high levels of sucrose (0.8 M, conventional protocol), or a combination of 0.4 M sucrose and 2 M glycerol. Recovery rates varied among genotypes (23-63%). The latter method reduced considerably the time needed for the cryogenic procedure by eliminating the pre-treatment with 0.8 M sucrose for 19 h prior to dehydration, as required by the conventional procedure.

Keywords: apices, cryopreservation, desiccation, encapsulation, Fragaria, germplasm



CryoLetters 26 (1), 25-32 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


María R. Fernández-Santos, Milagros C. Esteso, Ana J. Soler, Vidal Montoro
and José J. Garde*

Instituto de Investigación en Recursos Cinegéticos, IREC, (CSIC, UCLM, JCCM). Campus Universitario, 02071. Albacete, Spain.
*E-mail: (J. Garde)


With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition 22ºC (ambient temperature) and 5ºC on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G ~ EG ~ PG < DMSO (‘less than’ symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22ºC (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.  

Keywords: DMSO, ethylene glycol, glycerol, propylene glycol, red deer, sperm, toxicity



CryoLetters 26 (1), 33-44 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Thermal analysis of Garlic Shoot tips during a Vitrification Procedure

Haeng-Hoon Kim1, Ju-Won Yoon1, Jung-Bong Kim1, Florent Engelmann2, 3
and Eun-Gi Cho1*

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. (email for E.G. Cho:
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.


The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P < 0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P < 0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (> 80 %). There was a significant (P < 0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P < 0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.

Keywords: Allium sativum L., DSC, PVS3, vitrification



CryoLetters 26 (1), 45-54 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Graham Sherlock1, William Block2* and Erica E Benson1

1Plant Conservation Group, Conservation & Environmental Chemistry Centre, School of Contemporary Science, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, UK.
2British Antarctic Survey, Natural Environment Research Council, High Cross Madingley Road, Cambridge CB3 0ET, UK.
* corresponding author


The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination.  This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16ºC for 5 h decreased bead water content from ca.75 to 28% fresh weight (3.8 to 0.4 g.g-1 dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g.g-1 dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. Tg mid-point temperatures ranged from -78 to -51ºC (cooling) and from -88 to -54ºC (warming) [at cooling and warming rates of 10 and 5ºC min-1, respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16ºC). This reduces bead moisture content to a critical point (ca. 0.4 g.g-1 dry weight) at which  stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.

Keywords: Thermal analysis, cryopreservation, encapsulation-dehydration, desiccation, Ribes, glass transitions, vitrification



CryoLetters 26 (1), 55-64 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

In vitro evaluation of vascular endothelial and smooth muscle cell survival and apoptosis in response to hypothermia and freezing

Kristine N. Tatsutani1*, James D. Joye2, Renu Virmani3 and Michael J. Taylor4

1CryoVascular Systems, Inc., 160 Knowles Drive, Los Gatos, California, 95032. E-mail:
2 El Camino Hospital, Mountain View, California. 
3Armed Forces Insitute of Pathology, Washington, D.C.
4Organ Recovery Systems, Inc., Charleston, South Carolina.


The success of endovascular techniques such as balloon angioplasty and stenting in the treatment of atherosclerotic vascular disease has been limited by an aggressive proliferative response leading to neointimal hyperplasia and re-stenosis.  A new endovascular therapy combining cold treatment with balloon dilation has been proposed to prevent arterial re-stenosis. In order to evaluate the potential of this application, studies were conducted investigating the effects of hypothermia and freezing on human arteries at the cellular level.  Cultured arterial endothelial cells and smooth muscle cells were chilled or frozen under controlled thermal conditions.  The viability response of the cells was measured with a variety of assays quantifying necrosis, apoptosis, and cell proliferation. These data establish correlations between thermal conditions and the extent and nature of arterial freezing injury.  Arterial smooth muscle cells were found to be susceptible to freeze-induced apoptosis in a temperature range of –5 to –15ºC. Endovascular cryotherapy designed to induce apoptosis in arterial smooth muscle cells may limit neointimal formation and thereby improve the durability of conventional angioplasty.

Keywords: Arterial, endothelial cells, smooth muscle cells, angioplasty, apoptosis, necrosis



CryoLetters 26 (1), 65-72 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Olga I. Gordiyenko*, Yuliya E. Gordiyenko, Tamara P. Linnik and Evgen O. Gordiyenko

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


Erythrocyte membrane permeability coefficients have been determined for a series of amides by a method based on the physical and mathematical modelling of hypotonic haemolysis process. The results show that penetration of the substances occurs by two alternative ways – through aqueous pores formed by proteins and by the direct dissolving of the molecules in membrane lipids. This conclusion can be confirmed by the correlation analysis between permeability coefficients of native erythrocytes and those pre-incubated with the monosodium salt of p-chloromercuribenzenesulfonic acid (pCMBS), and the partition coefficients of the substances in hydrophilic-hydrophobic phases. Penetration of substances through hydrophilic channels is limited by the sterical factor and diameter in particular. Permeability coefficients for erythrocytes pre-incubated with pCMBS increase in an accordance with the rise of the partition coefficients with correlation coefficient of 0.94, thereby indicating a lipid route of permeation of molecules through erythrocyte membranes.

Keywords: human erythrocyte, permeability coefficients, amides.

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