Abstracts: CryoLetters 26 (3), 2005

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

CryoLetters 26 (3), 139-146 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification

T. Hirano1, K. Ishikawa2 and M. Mii1*

1 Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-City, Chiba, 271-8510, Japan.
2 Japan Horticultural Production and Research Institute, 2-5-1 Kamishiki, Matsudo-City, Chiba, 270-2221, Japan.
*For correspondence:


Ponerorchis graminifolia var. suzukiana is a terrestrial orchid that is an endangered species native to Japan, and it germinates more readily in immature seeds than in mature seeds. To preserve this orchid, an efficient protocol was established for the cryopreservation of immature seeds of P. graminifolia var. suzukiana. When immature seeds of 6 weeks after pollination, which showed higher germination and protocorm formation than mature seeds, were precultured on New Dogashima (ND) medium with 0.3M sucrose for 3 days and cryopreserved by vitrification method (treated with PVS2 for 60 min), the viability after preservation as assessed with 2,3,5-triphenyltetrazolium chloride staining test was about 86%. Immature seeds thus treated showed equal rates of germination and protocorm formation to the untreated control immature seeds, and they developed into normal plantlets on ND medium.

Keywords: cryopreservation, endangered species, immature seeds, Ponerorchis graminifolia var. suzukiana, vitrification



CryoLetters 26 (3), 147-158 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

The Effect Of cell size distribution on predicted osmotic responses of cells

H. Y. Elmoazzen1,2, C. C. V. Chan1, J. P. Acker2, J. A. W. Elliott1, L. E. McGann2

1Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada, T6G 2G6
2Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2R8


An understanding of the kinetics of the osmotic response of cells is important in understanding permeability properties of cell membranes and predicting cell responses during exposure to anisotonic conditions.  Traditionally, a mathematical model of cell osmotic response is obtained by applying mass transport and Boyle-van’t Hoff equations using numerical methods. In the usual application of these equations, it is assumed that all cells are the same size equal to the mean or mode of the population. However, biological cells (even if they had identical membranes and hence identical permeability characteristics – which they do not) have a distribution in cell size and will therefore shrink or swell at different rates when exposed to anisotonic conditions.  A population of cells may therefore exhibit a different average osmotic response than that of a single cell. In this study, a mathematical model using mass transport and Boyle-van’t Hoff equations was applied to measured size distributions of cells. Chinese hamster fibroblast cells (V-79W) and Madin-Darby canine kidney cells (MDCK), were placed in hypertonic solutions and the kinetics of cell shrinkage were monitored. Consistent with the theoretical predictions, the size distributions of these cells were found to change over time, therefore the selection of the measure of central tendency for the population may affect the calculated osmotic parameters.  After examining three different average volumes (mean, median, and mode) using four different theoretical cell size distributions, it was determined that, for the assumptions used in this study, the mean or median were the best measures of central tendency to describe osmotic volume changes in cell suspensions.

Keywords: osmotic kinetics, cell volume distributions, membrane hydraulic conductivity, hamster fibroblasts, canine kidney cells, measures of central tendency



CryoLetters 26 (3), 159-168 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Takashi Suzuki1*, Daisuke Kami1, Katsuji Oosawa1, and Locksley E. McGann2

1Department of Horticulture and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. 
2Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T7G 2R8, Canada


To clarify the mechanism of reduced volume expansion-related cryoprotection changes in solution volume during freezing using several types of cryoprotectant were investigated. The effect of each cryoprotectant solution on the survival of asparagus nodal segments cooled slowly (0.5˚C/min) to -40˚C was also examined. The ratio of the volume at -40˚C to the volume at +20˚C was used as an index for expansion, calculated as a ratio of the density at +20˚C to the density at -40˚C. Distilled, deionized water showed the largest volume change at a ratio of 1.094. The ratio gradually decreased with an increase in the molar concentration of cryoprotectant, with the magnitude of the change dependent on the nature of the cryoprotectant. Raffinose was the most effective in reducing volume expansion when compared with other cryoprotectants at a same concentration. Raffinose exhibited greatest cryoprotection in asparagus tissue at 0.6 M where the solution became saturated. Dimethyl sulfoxide (Me2SO) at 1.69 M had the largest effect on cryoprotecting asparagus tissue. Furthermore, Me2SO was also the most effective in reducing volume expansion among the group of cryoprotectants permeable to the plasma membrane. It is concluded that cryoprotection in tissues was closely related to reduced volume expansion especially at low concentration (≤1.0 M). Cryoprotectants of impermeable sugar group lost their cryoprotective effect at >1.0 M, which may due to severe dehydration and cell damage occurred in hypertonic solution. Useful cryoprotectants should be furnished with high ability of reducing volume expansion during freezing as well as low toxicity and high permeability for cells.

Keywords: Asparagus officinalis L., cryopreservation, freezing injury, ice formation, slow freezing



CryoLetters 26 (3), 169-184 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Pablo J. Giraudi, Luciana L. Almada, María E. Mamprin, Edgardo E. Guibert1, Graciela Furno2 and Joaquín V. Rodriguez.

Farmacología, Departamento de Ciencias Fisiológicas and 1Biología Molecular, Departamento de Ciencias Biológicas, 2Estadística, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531-S2002LRK Rosario, Argentina.


A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence-Fd) and after (total fluorescence-Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.

Keywords: UW, Propidium Iodide, hepatocytes, cold preservation, subzero nonfreezing preservation, cell viability, fluorometric assay



CryoLetters 26 (3), 185-192 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Maurizio Lambardi1*, Anna De Carlo1 and Maurizio Capuana2

1 Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze), Italy.
2 Istituto di Genetica Vegetale, sezione di Firenze, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze), Italy.
* For correspondence:


An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at –196°C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45°C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.

Keywords: horse chestnut, Aesculus hippocastanum, cryopreservation, vitrification, somatic embryogenesis.



CryoLetters 26 (3), 193-200 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jun Li1, Tse-Chao Hua1*, Xue-Lian Gu1, Yu Ding1, Ming Luo1, Hong-Hai Xiao1, Zhi-Jiang Wu2, Lv-Rong Meng2, Qi-Rong Gao2 and Jian Chen2

1Institute of Cryomedicine and Food Refrigeration, University of Shanghai for Science and Technology, No.516 Jun-Gong Road, 200093, Shanghai, China
2Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, No.320 Yo-Yang Road, 200031, Shanghai, China
*corresponding author: Tse-Chao Hua, Institute of Cryomedicine and Food Refrigeration, Tel: 86-21-65685291, Fax: 86-21-65685291


The research on haematopoietic stem cells of human cord blood has become more important recently. At present, cord blood is mainly preserved at ultra-low temperatures. In the former study, we compared the effects of preserving mononuclear cells (MNC) and whole human cord blood by freeze-drying. This time, a further study was conducted on freeze-drying mononuclear cells. Samples in the presence of PVP, sucrose, mannitol and FBS were firstly frozen to -38°C. Afterwards, they were vacuum-dried at a selected shelf temperature of -30°C for the main drying stage, and then vacuum-dried at 15°C for the second drying stage. The entire time of freeze-drying process was 41 hours. Samples were stored at room temperature for 7 days prior to evaluation. Subsequently, the dried samples were resuspended in an isotonic phosphate-buffered saline solution.
The residual moisture content was 6.5±0.87%. The recovery of the cells was tested by a haemacytometer, and the numerical cell count recovery of rehydrated MNC increased by 8%. Morphology of the fresh and rehydrated MNC was analyzed respectively using standard light microscopy, scanning electron microscope and transmission electron microscope. The results showed that karyons changed and cytoplasm decreased after rehydration, but it is still unknown that whether these changes will influence the proliferative ability of the stem cells.

Keywords: freeze-drying, human cord blood, MNC, lyoprotectant, TEM



CryoLetters 26 (3), 201-212 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Comparison of Cryopreservation Techniques for Long-Term Storage of Ash (Fraxinus excelsior L.)

Katja Schoenweiss1,2,*, Andreas Meier-Dinkel1 and Rüdiger Grotha2

1Lower Saxony Forest Research Institute, Department of Forest Genetic Resources, Forstamtstrasse 6, 34355 Staufenberg, Germany.
2Institut für Biologie, Universität Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany


The main purpose of this study was to develop a cryopreservation protocol for ash and to highlight the importance of testing different clones and plant material of different ontogenetic states. In vitro-grown ash (Fraxinus excelsior L.) shoot tips were successfully cryopreserved following optimization of the PVS2-vitrification protocol. Pretreatment conditions were optimized and three cryopreservation techniques (encapsulation/dehydration, PVS2-vitrification and encapsulation/vitrification) were tested one after another. PVS2-vitrification proved to be the most suitable technique. In vitro-grown shoot tips of ash were successfully cryopreserved with a mean regrowth of 73 % for juvenile clones and 67 % for selected mature trees. The optimum preculture conditions and the initial protocol were: 10 days cold hardening, preculture for 2 days on medium with 0.8 M glycerol, incubation in 2 M glycerol solution for 20 min at 22°C followed by PVS2 for 25 min at 0°C on ice and direct immersion in liquid nitrogen. Warming was carried out in 43°C water for 1 min followed by 22°C water for 10 sec. The encapsulation/dehydration method was not successful for shoot tips of F. excelsior because the shoots were sensitive to osmotic dehydration. The encapsulation/vitrification method resulted in a mean regrowth of only 16 %. PVS2 vitrification can now be used to store important ash germplasm of either juvenile or mature trees.

Key words: conservation, in vitro, juvenile, mature, forest tree, vitrification, encapsulation

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