Abstracts: CryoLetters 22 (5), 2001

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

CryoLetters 22, 273-276 (2001)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Martin Holmstrup* and Heidi Sjursen

National Environmental Research Institute, Department of Terrestrial Ecology, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark
Phone +45 8920 1400; Fax +45 8920 1414; *e-mail:


The tolerance of freezing and associated accumulation of cryoprotectants was studied in an Arctic population of the enchytraeid Fredericia ratzeli. At -3 and -5ºC specimens readily froze when slowly cooled in contact with moist soil. A small fraction of the animals (10-20 %) survived internal ice formation under these circumstances. Frozen specimens had elevated glucose concentrations as compared to unfrozen control animals acclimated at 0ºC. In a fraction of the animals, equal to the fraction surviving freezing, a high concentration of glucose was detected. The highest values amounted to ca. 150 µg mg-1 dry weight, corresponding to ca. 270 mmoles L-1. It is argued that the physiology of freeze tolerance in this enchytraeid resembles the physiology described for freeze tolerant earthworms and frogs.

Keywords: Freeze tolerance; enchytraeid; terrestrial oligochaete; glucose; inoculative freezing.



CryoLetters 22, 277-284 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Cryopreservation of Kiwi shoot tips

Yongjie Wu1*, Yanhua Zhao1, Florent Engelmann2, 3 and Mingde Zhou4

1Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Hebei 066600, Changli Town, China. (Email:
2IPGRI, Via dei Tre Denari, 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
3IRD, BP 5045, 34032 Montpellier Cedex 01, France (present address).
4IPGRI, East Asia Office c/o CAAS, 30 Bai Shi Qiao Road, Beijing100081, China.


In this study, the effect of various factors, including the physiological state of mother-plants and parameters of the encapsulation-dehydration protocol (cryoprotective treatment, freezing protocol) on the regrowth of shoot tips of kiwi in vitro plantlets was studied. The optimal protocol established was the following: shoot tips were sampled on mother-plants after a one month cold-acclimation period at 5°C. For preculture, which was performed at 5°C, encapsulated shoot tips were transferred at 24-hour intervals on solid media with increasing sucrose concentrations, from 0.5 to 1.0M. Beads were then desiccated to 26% moisture content (fresh weight basis), prefrozen from 0°C to -40°C at 0.2°C/min and immersed rapidly in liquid nitrogen. No differences were noted between in vitro plantlets produced from cryopreserved and control shoot tips as regards leaf color, average height, multiplication rate and peroxidase zymogram pattern. Apices of 3 kiwi accessions were frozen using the above protocol with regrowth percentages ranging between 22 and 56%.

Keywords: Actinidia spp.; cryopreservation; shoot tip; encapsulation-dehydration; preculture; freezing protocol.



CryoLetters 22, 285-292 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Effect of Dextran MOlecular weight on Protein stabiliation during freeze-drying and storage

Wendell Q. Sun* and Paul Davidson

Department of Biological Sciences, National University of Singapore, Singapore 119260. *Present address: LifeCell Corporation, Branchburg, NJ 08876, USA (


The effect of dextran molecular weight on structural stability of freeze-dried products and protein stability in amorphous matrices was investigated during storage at elevated temperatures.  Glucose-6-phosphate dehydrogenase (G6PDH) was freeze-dried in 10% (w/v) dextrans of five molecular weights (12 kD, 42 kD, 71 kD, 512 kD and 2000 kD) to residual water content of 0.027  0.004 g/g dry mass. The molecular weight of dextrans affected the glass transition temperature (Tg) of freeze-dried products and the recovery of enzyme activity after freeze-drying. As the molecular weight of dextrans increased from 12 kD to 2000 kD, the Tg increased from 100 C to 120 C, whereas the recovery of protein activity decreased from 85 4 % to 70  5%. The inactivation of freeze-dried protein during storage followed a bi-phasic first-order kinetics. The stability of amorphous matrices and protein increased significantly as the molecular weight increased from 12 kD to 512 kD.  However, at a higher molecular weight (2000 kD), the stability was reduced.  In a separate experiment, the stability of dried dextran/protein samples was studied during heating from 30 C to 99 C at 0.2 C/min and subsequent incubation at 99 C. Dextran with an average molecular weight of 512 kD was again found to provide the best protection. Mechanisms that cause the differences in protein stability among different molecular weight dextrans remain unclear.

Keywords: dextrans, freeze-drying, glass transition, molecular weight, protein stabilization, structural collapse, thermal inactivation.



CryoLetters 22, 293-298 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


A. V. Popova* and M. R. Busheva

Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev str. bl. 21, 1113 Sofia, Bulgaria,  *


The mechanism of action of the osmoregulatory substances glycine betaine and glycerol in the course of the freeze-thaw cycle was studied. Photochemical activity of isolated thylakoid membranes was effectively protected during freezing by both solutes by preventing dissociation of the peripheral cold labile proteins - the water splitting system and plastocyanin. It is suggested that the cryoprotective effect of glycine betaine and glycerol is based on a mechanism similar to the well documented stabilization of complex enzymes against dissociation into subunits at high salt concentrations. Specific solute-membrane interactions additionally contribute to the observed cryoprotective effect. The binding of the molecules of glycine betaine and glycerol is limited to the water lipid interface of thylakoid membranes.

Keywords: thylakoid membranes, photochemical activity, cryoprotection, lipid fluidity, solute-membrane interaction.



CryoLetters 22, 299-310 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Nelly Odintsova*,  Konstantin Kiselev**, Nina Sanina** and Edward Kostetsky**

*Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Vladivostok, Russia 690041. E-mail:
**Far Eastern State University, Vladivostok, Russia 690090.


Primary cell cultures obtained from somatic and larval tissues of bivalve molluscs and from embryos of sea urchins were frozen to –196ºC by two-step freezing using 10% dimethyl sulfoxide (DMSO) or/and trehalose (3-30mg/ml) as cryoprotectants. We estimated both cell viability and the RNA synthetic activity after freeze-thaw. Total lipid extracts from the tissues of echinoderms examined as possible cryoprotective agents demonstrated a weak cryoprotective capacity. Mussel lipid extract was found to possess a considerable cryoprotective activity. Cryoprotective capacity of tested lipids correlated with their thermotropic behaviour. DMSO + trehalose combination was shown to be a favourable cryoprotectant and sea urchin blastula cells the most freezing-tolerant cells.

Keywords: Cryopreservation, primary cell culture, mollusc, sea urchin, trehalose, total lipids, phase transition.



CryoLetters 22, 311-320 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Kazuhito KAJIWARA*, Akihito MOTEGI and Norio MURASE1

Department of Biosciences, Faculty of Science and Engineering, Teikyo University of Science and Technology, 2525 Yatsusawa Uenohara-machi, Kitatsuru-gun, Yamanashi, 409-0193, JAPAN
1Department of Biotechnology, College of Science and Engineering, Tokyo Denki University, Hatoyama-cho, Hiki-gun, Saitama, 350-0394, JAPAN


The freeze-thawing behaviour of highly concentrated aqueous alkali chloride-glucose systems was investigated by differential scanning calorimetry (DSC). In the aqueous NaCl-glucose solution system, single or double glass transitions followed by the corresponding devitrification exotherms were observed during rewarming. In the aqueous KCl-glucose solution system, on the other hand, a single glass transition followed by an exotherm was observed during rewarming.  The presence of double glass transitions observed for a certain composition of the aqueous NaCl-glucose solution was taken as an evidence for the liquid-liquid immiscibility at low temperatures. Two kinds of crystallisation accompanied by exotherms during rewarming were identified by X-ray diffraction as ice and ice/NaCl·2H2O, or ice/KCl eutectic component.

Keywords: Freeze-thawing, Low-temperature DSC, Aqueous alkali chloride-glucose systems, Double glass transition, Solution heterogeneity, X-Ray diffraction.



CryoLetters 22, 321-328 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Yanhua Zhao1, Yongjie Wu1*, Florent Engelmann2, 3 and Mingde Zhou4

1Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Hebei 066600, Changli Town, China. (Email:
2International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari, 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
3Institut de recherche pour le développement (IRD), BP 5045, 34032 Montpellier Cedex 01, France (present address).
4International Plant Genetic Resources Institute (IPGRI), East Asia Office c/o CAAS, 30 Bai Shi Qiao Road, Beijing 100081, China.


Axillary buds sampled from in vitro plants of four grape varieties could withstand cryopreservation using the encapsulation-dehydration technique. Regrowth percentages ranged between 15 and 40%. Buds were encapsulated in alginate beads containing 0.5M sucrose, precultured at 5°C on media containing daily increasing sucrose concentrations from 0.1 to 1.0M, then desiccated to 21% moisture content (fresh weight basis) and frozen slowly (prefreezing to -40°C at 0.2°C/min followed by immersion in liquid nitrogen). The physiological state of the in vitro mother-plants and the freezing procedure employed dramatically influenced the results. Regrowth after cryopreservation was achieved only when buds were sampled from mother-plants that had been kept without subculture for 3 to 4 months. An additional one-month period of cold-acclimation of mother-plants at 5°C and a two-step freezing procedure improved growth recovery. The multiplication rate of shoots produced from cryopreserved buds was lower than that of controls during the first subcultures after thawing. However, the rooting ability of control and cryopreserved plantlets became similar from the fourth subculture onwards.

Keywords: grape; Vitis vinifera; cryopreservation; encapsulation-dehydration; cold-acclimation; freezing rate.



CryoLetters 22, 329-336 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Ken Muldrew*, Sherri Liang, Zhihong Liu, and Richard Wan

Department of Surgery, Department of Civil Engineering, University of Calgary, Calgary, Canada
*Joint Injury and Arthritis Research Group,Heritage Building for Medical Research, 3330 Hospital Dr. NW, Calgary, Alberta  T2N 4N1, Canada.
 Tel. (403) 220-5976 Fax (403) 270-0617


The present study is part of a program to optimize the cryopreservation ligament and tendon allografts for the biological remodeling that occurs following transplantation. The osmotic behavior of fibroblasts from the medial collateral ligament (MCL) and the patellar tendon (PT) of new zealand white rabbits was measured to obtain the hydraulic conductivity (and its temperature dependence) as well as the osmotically inactive volume. MCL fibroblasts were found to have an isotonic cell volume of 3800 m3, an osmotically inactive volume of 1300 m3, and a hydraulic conductivity of 0.56 m/min/atm at 20°C with an activation energy of 10.9 kcal/mol. PT fibroblasts were found to have an isotonic cell volume of 6300 m3, an osmotically inactive volume of 2000 m3, and a hydraulic conductivity of 0.71 m/min/atm at 20°C with an activation energy of 10.1 kcal/mol.

Keywords: Osmotic behavior, Permeability, Ligament, Tendon, Fibroblast.

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