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Abstracts: CryoLetters 27 (6), 2006

CryoLetters 27 (6), 333-340 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


T.C. Hawes

Department of Biosciences, University of Birmingham, Edgbaston Birmingham B15 2TT, U.K. E-mail:


Cryobiologists have traditionally assumed that the temperature of crystallisation (Tc) or supercooling point (SCP) of a chill-tolerant insect is not a stochastic event, i.e. that it is a biologically meaningful indicator of phenotypic characteristics, be they exogenous influences (e.g. acclimation/acclimatization) or endogenous factors (e.g. life history stage, moult state). Recent work by Wilson et al. (11) has suggested that SCPs – at least in non-biological samples – are more stochastic than previously thought. Here, this question is tested indirectly by the repetitive freezing of individuals of the Antarctic springtail, Cryptopygus antarcticus. The springtails were each supercooled ten times in succession to determine their re-crystallisation temperatures (Trc). SCPs were found to be deterministic i.e. related to their initial Tc. Despite the mortality of re-crystallised samples, 70% showed <1°C difference between Tc and Trc1 and 95% showed <5°C difference. Tc and Trc1 were significantly correlated. Variability in re-crystallisation temperatures is hypothesised to be predominantly the result of differences in nucleator content and changes in body fluid osmolality during the experimental exposures. Factors affecting the relative variability of SCPs are discussed.

Keywords: supercooling point, re-crystallisation, nucleation, Collembola, determinism



CryoLetters 27 (6), 341-352 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Li Luo, Yi Pang, Qijin Chen and Guanghong Li*

State Key Laboratory of Biocontrol & Institute of Entomology, Sun Yat-sen University, No.135 Xin Gang Xi Road, Guangzhou, Guangdong Province, P.R.China. 510275
*Corresponding author, e-mail:


Genetic devolution, genetic drift and contamination are all threats to maintain germplasm stability during mass rearing of many insects. Cryopreservation of beet armworm (Spodoptera exigua) embryos was studied to provide information to improve mass rearing. A series of experiments was conducted on late-stage embryos (45-48 h at 27°C) of the beet armyworm, which included evaluation of cryoprotectants (CPAs), their toxicity and glass-forming tendency and optimization of experimental procedures. The results showed that ethylene glycol (EG) was the best CPA with comparatively low toxicity compared to the other six CPAs tested (methanol, 1,3-propanediol, glycerol, 2-amino-1-ethanol, 3-amino-1-propanol 3-methoxy-1 and 2-propanediol). The highest hatching rate of 8.8% was attained after freezing with a 3-step loading procedure and a 1-step unloading procedure, but the hatched larvae from frozen-thawed embryos did not actively feed and could not develop to a later stage. This was attributed to injuries from freezing in late stage embryos of S. exigua which had formed midguts.

Keywords: cryopreservation, vitrification, Spodoptera exigua, insect embryo, cryoprotectants, ethylene glycol



CryoLetters 27 (6), 353-360 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Leigh E. Towill, Remi Bonnart and Gayle M. Volk*

USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA


Development of a successful shoot tip cryopreservation method for Arabidopsis thaliana L. will enable researchers to use molecular tools to study processes important for successful cryopreservation in this model organism.  We demonstrate that Arabidopsis can be successfully cryopreserved using either plant vitrification solution 2 (PVS2) or plant vitrification solution 3 (PVS3) as cryoprotectants prior to rapidly cooling shoot tips in liquid nitrogen (LN).  Shoot tip regrowth after PVS2 cryoprotectant treatment was improved after cold acclimation treatments of 8 or 18 days.  All of the shoots tips regrew after LN exposure when cryoprotected with PVS3 for 60 min at 22 °C. In addition, shoot tips could be cryopreserved using a two-step cooling procedure with PGD (polyethylene glycol-glucose-dimethyl sulfoxide) as a cryoprotectant.  The high levels of shoot formation after LN exposure of Arabidopsis shoot tips makes this a desirable system in which molecular tools can be used to examine how alterations in biochemical, metabolic and developmental processes affect regrowth after cryoprotective treatments.

Keywords: vitrification, two-step cooling, cryoprotectant, liquid nitrogen, cold acclimation



CryoLetters 27 (6), 361-368 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Peng-Fei Yang1, Tse-Chao Hua1*, Jing Wu1, Zhao-Hua Chang1, Hsiao-Chien Tsung2 and Yi-Lin Cao2

1Institute of Cryomedicine and Food Refrigeration, Shanghai University of Science and Technology, No.516 Jun-Gong Road, 200093, Shanghai, China.
* Tel: 86-21-65685291. Fax: 86-21-65685291. E-mail:
2Laboratory of Tissue Engineer, The 9th People' Hospital, Shanghai 2nd Medical University, No.639 Zhi Zao Ju Road, 200011, Shanghai, China


Human embryonic stem (ES) cells have far-reaching applications in the areas of tissue engineering, regenerative medicine, pharmacology and basic scientific research. Although the culture conditions can maintain the human ES cells in an undifferentiated state for a transient period, spontaneous differentiation has also been observed during the routine culturing of ES cells. However, the maintenance of ES cells in the undifferentiated, pluripotent state for extended periods of time will be required in many areas of scientific research. Cryopreservation is a technology with potentially far reaching implication for the development and widespread use of such cell lines. This study was undertaken to develop and optimize a protocol for cryopreservation of human ES cells through programmed cooling. The effects of the seeding temperature, the cooling rate and the sub-zero temperature to which the samples were cooled before plunging into liquid nitrogen(the terminal temperature), all significantly affected the recovery of cryopreserved ES cells. After studying these factors, an improved protocol was obtained: the sample was cooled from 0°C to –35°C at a cooling rate of 0.5°C /min , with seeding was set at –10°C, before being plunged immediately into the liquid nitrogen. Using this protocol, 9 of 11 colony fragments survived freezing and thawing and could be cultured for prolonged periods. They retained the properties of pluripotent cells, had a normal karyotype and showed histochemical staining for alkaline phosphatase.

Keywords: cryopreservation , human embryonic stem cell



CryoLetters 27 (6), 369-374 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Baolin Liu1* and John J. McGrath2

1Institute of Cryobiology, Shanghai University of Science and Technology, Shanghai 200093, China.
*Corresponding author: Tel: 86-21-65688765. Email:
blliuk@163.com2Department of Aerospace and Mechanical Engineering, University of Arizona, Tucson, AZ, 85721, USA


Understanding the ultra-structural response of cells to the cryopreservation process is important for designing cryopreservation strategies for cells and tissues. Cell-cell interaction and cell-scaffold interactions alter cryopreservation response and, in turn, the cellular structures involved in adhesion and intercellular contact are possible targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments (F-actin), focal adhesions (vinculin) and gap junctions (connexin-43) of murine osteoblasts attached to hydroxyapatite (HA) discs and plastic coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the actin filaments of dead cells, while those of live cells experienced little change. Vinculin and connexin-43 structures were rarely seen in dead cells, while a portion of vinculin (8.14  2.27%) and connexin-43 (21.7  4.7%) structures remained in live cells. These results suggest that focal adhesions and gap junctions may support cell robustness during cryopreservation. The present study contributes to our knowledge of the damage mechanisms associated with attached cells during a freezing process.

Keywords: osteoblasts, actin filaments, focal adhesions, gap junctions, two-step freezing



CryoLetters 27 (6), 375-386 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Raj Kumari Sanayaima1, Amandeep Kaur1, Anuradha Agrawal2
and Shashi Bhusan Babbar1*

1Department of Botany, University of Delhi, Delhi-110 007, India.
2National Bureau of Plant Genetic Resources, New Delhi - 110012, India


Cryopreservation of in vitro axillary shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree, was investigated. Axillary buds (c. 1mm in length) excised from 4-week-old in vitro cultures, were pre-cultured on liquid MS medium supplemented with 0.4 M sucrose for 16 h. These were incubated in 2 M glycerol + 0.4 M sucrose for 20 min at 25°C before being dehydrated with PVS2 solution for 40 min The dehydrated shoot tips were directly immersed in LN. Following cryopreservation and after rapid warming at 40°C, shoot tips were quickly washed with MS + 1.2 M sucrose solution for 20 min and then plated on top of filter paper placed on MS medium supplemented with 0.1 mg l-1 BAP, kept in darkness for one day followed by placement of shoots directly on the medium and incubation in darkness for a day more, before transfer of cultures to light. Average survival in terms of normal shoot formation after 4 weeks of plating was 56.6%. The rescued shoot tips were bulked up by subsequent nodal cultures and when put onto 0.02 mg l-l NAA showed a rhizogenic response. Thus, in vitro-grown shoot tips of Crateva nurvala were successfully cryopreserved following the optimization of the PVS2-vitrification protocol.

Keywords: Crateva nurvala, cryopreservation, axillary buds, vitrification



CryoLetters 27 (6), 387-399 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


T. F. Sørensen1*, C-H.C. Cheng2 and H. Ramløv1

1Dept Life Sciences & Chemistry Roskilde University DK4000 Roskilde Denmark
* Corresponding author email:
2Dept. Animal Biology, University of Illinois at Urbana-Champaign, Urbana IL, 61801, USA


The European eelpout Zoarces viviparus is a common inhabitant in the coastal areas of the eastern Atlantic Ocean and the Baltic region. At least 3 different antifreeze proteins were purified from Z. viviparus serum but more isoforms are most likely present. Two antifreeze proteins with molecular weights of approx. 6.5-7 kDa were characterised and found to share high similarity to the type III antifreeze proteins found in other members of the family Zoarcidae. The antifreeze activity of Z. viviparus antifreeze proteins is concentration dependent and showed a saturation effect when the protein concentration reached 30mg•ml-1 (crude serum) and 8mg•ml-1 (partly purified serum) respectively. Further the antifreeze activity was found to be dependent of the buffer osmolality resulting in increasing thermal hysteresis when buffer osmolality was raised from 0 to 1M.

Keywords: Antifreeze proteins, Zoarces viviparus, Zoarcidae, purification, characterisation.

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