Abstracts: CryoLetters 25 (4), 2004

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

CryoLetters 25, 239-240 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK






CryoLetters 25, 241-254 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


L.L. Kuleshova1, 2,*, X. W. Wang3,4, Y.N. Wu1, Y. Zhou2, H. Yu1

1Department of Physiology, National University of Singapore,
2National University Medical Institutes, National University of Singapore,
3 Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, Singapore.
4Department of General Surgery, First Hospital of Xiang Ya Medicine College, Zhong Nan University, Changsha Hunan, China.
*To whom correspondence should be addressed: National University Medical Institutes, National University of Singapore, Block MD11, 10 Medical Drive, Singapore 117597,


We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400C/min and above) and three warming rates (650C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.

Keywords: cryopreservation, encapsulated hepatocytes, vitrification, ethylene glycol, double straw.



CryoLetters 25, 255-264 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


A.V. Popova*, and M.Y. Velitchkova

Institute of Biophysics, Bulgarian Academy of Asciences, Acad. G. Bonchev str. Bl. 21, 1113 Sofia, Bulgaria,


The extent of freezing damage of the photosynthetic apparatus of isolated thylakoid membranes, control and after modification of their membrane fatty acid acyl chain order by cholesterol and benzyl alcohol, was studied. The photochemical activity of photosystems I and II and the energy transfer between the main pigment protein complexes had been determined. Cholesterol-treated membranes are less susceptible to freezing damage, expressed by minor changes of the photochemical activity and retaining the 77K fluorescent characteristics. Benzyl alcohol incorporation enhanced the degree of freezing damage. The photochemical activity of both photosystems was severely decreased (by 80%) and considerable changes in the fluorescent properties were observed, mainly in the pigment pool associated with Photosystem I. The effects of different freezing media (artificial stroma medium, trehalose, glycine betaine and NaCl) were compared in respect to the maintaining of the activity of photosynthetic apparatus.

Keywords: thylakoid membranes, freezing damage, cholesterol, benzyl alcohol, lipid acyl chain order.



CryoLetters 25, 265-272 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


G. J. Morris* and H. E. Richens

Asymptote Ltd., St Johns Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK
*To whom correspondence should be addressed; email:


Simple, reproducible, methods of achieving rapid rates of cooling in the range 100°C min-1 to 1000°C min-1 are described for straws and cryovial.  These methods use the direct contact of straws or cryovials with pre-cooled granules or plates as the heat sink. Liquid nitrogen may be adsorbed into suitable material, for example activated charcoal, zeolites and molecular sieves as the matrix to achieve rapid cooling. Controllable rapid rates of cooling may also be attained by using non-adsorbants. The rate of cooling may be modified by changing the adsorbant material, the size of the adsorbant granules and the temperature of the adsorbant. 

Keywords: Cryopreservation, rapid cooling rates, CryoSEM



CryoLetters 25, 273-285 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Slađan Z. Pavlović1*, Dragana Belić1, Duško P. Blagojević1, Ratko M. Radojičić2, Radoslav V. Žikić3, Zorica S. Saičić1, Gordana Grubor-Lajšić4 and Mihajlo B. Spasić1

1 Institute for Biological Research “Siniša Stanković”, Department of Physiology, 29 Novembra 142, 11060 Belgrade, Serbia and Montenegro (email:
2 Faculty of Biology, Institute of Biochemistry and Physiology, University of Belgrade, Studentski Trg 16, 11000 Belgrade, Serbia and Montenegro.
3 Faculty of Sciences, Institute of Biology, University in Kragujevac, Radoja Domanovića 12, Kragujevac, Serbia and Montenegro.
4 Faculty of Sciences, Institute of Biology, University in Novi Sad, Dositeja Obradovića 1,  Novi Sad, Serbia and Montenegro.


The activity of cytosolic antioxidative defence enzymes in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) were compared in winter and spring in the Adriatic Sea.. Activity of antioxidative enzymes is functionally organized due to metabolic demands: analyses of variance and correlation analysis revealed tissue- and seasonal- specific organization of antioxidative enzymes. In winter GST activity increased in both tissues compared with spring. At the same time decreased GSH-Px and GR activities were observed and this effect was more pronounced in liver then in white muscle. From correlation analyses it is concluded that the antioxidative components correlate, but the composition of the antioxidative defence system is different in respect to season and tissue. This means that the antioxidative defence system reorganizes its structure due to oxidative demands and to protect the tissues against reactive oxygen species and to establish homeostasis. Discriminant analyses separated groups according to the complete organization of individual components of the system very well and identified individual components (CAT, GST and GR) which contribute most to the differences. Statistical differences were observed between enzyme activities in tissues (liver and muscle) in both winter and spring, and between seasons (winter and spring) for liver tissue only. Since environmental parameters, such as temperature and oxygen concentration in the sea differ with season, we conclude that in this species the tissues examined expressed their antioxidative defence systems in different ways in respect of external/environmental conditions. We propose that tissue- and seasonal- specific levels of antioxidant enzyme activities should be considered in the interpretation of data from future biomonitoring field studies, especially in relation to low temperature.

Keywords: Antioxidant defence enzymes, biomonitoring, season, temperature, thinlip gray mullet, Liza ramada



CryoLetters 25, 287-300 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of sea urchin (Evechinus chloroticus) SPERM

Serean L. Adams1, Paul A. Hessian2* and Philip V. Mladenov1

1Department of Marine Science and Leukocyte Inflammation Research Laboratory,
2Department of Physiology, University of Otago, P O Box 56, Dunedin, New Zealand
*Corresponding Author. Present Address: Leukocyte Inflammation Research Laboratory, Department of Physiology, Dunedin School of Medical Sciences, University of Otago, P. O. Box 913, Dunedin, New Zealand


A method was developed for cryopreserving sperm of the sea urchin, Evechinus chloroticus. Sperm fertilisation ability, mitochondrial function and membrane integrity were assessed before and after cryopreservation. Highest post-thaw fertilisation ability was achieved with lower concentrations (2.5%-7.5%) of dimethyl sulphoxide (DMSO). In contrast, post-thaw mitochondrial function and membrane integrity were higher for sperm frozen in intermediate and high DMSO concentrations (5%-15%). Surprisingly, some sperm frozen in seawater only, without DMSO, were able to survive post-thawing, although the fertilisation ability (106 sperm/ml; ~50% fertilisation), mitochondrial function and membrane integrity of these sperm were notably lower than of sperm frozen with DMSO (106 sperm/ml; 2.5%-7.5% DMSO; >85% fertilisation) at the concentrations tested. Amongst sperm from individual males, fertilisation ability varied before and after cryopreservation for both males frozen with and without cryoprotectant. Specific differences among males also varied. Sperm mitochondrial function and membrane integrity was similar among males before cryopreservation but differed considerably after cryopreservation. Cryopreserved sperm were able to fertilise eggs and develop to pluteus stage larvae. This study has practical applications and will provide benefits such as reduced broodstock conditioning costs, control of parental input and opportunities for hybridisation studies.

Keywords: cryopreservation, sea urchin, sperm, freezing, marine invertebrate



CryoLetters 25, 301-306 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Effects of Hypothermia on Ischaemic Depolarisation in Brain : A comparison of responses in Rat and Gerbil

Barry Fuller1 and Edward Proctor2

1University Department of Surgery, Royal Free & UC Medical School, Pond Street, London NW3 2QG &
2RCS Unit of Biophysics, Institute of Child health, Guilford Street, London WC1N 1EH, UK.


The latency of development of ischaemic depolarization (LID) has been used to compare the relative effects of different levels of hypothermia on ischaemic responses in brains of rat and gerbil, using a model with imposed complete cessation of cerebral blood flow (CBF=0). At temperatures reducing from 37oC to 20oC, the LIDs were consistently shorter in the gerbil than in the rat. For example, at 37oC the LID in the gerbil was 0.71±01.2 min, and in the rat, 1.37±0.02 min respectively (P<0.01), whilst at 20oC, the values were 5.48±0.25 min and 7.30±0.76 min (P<0.01). However, the relative effects of hypothermia on each species were similar (by linear regression with slopes of -0.29 and -0.35 min/oC in the two species). There may be underlying differences in brain biophysics or structure between species, but in spite of this, applied hypothermia still imposes a similar depression on the development of ischaemic damage. Both models may thus be used in studies of brain hypothermia as long as the intrinsic differences are appreciated.

Keywords: Hypothermia; brain ischaemia; latency of ischaemic depolarization; comparison of rat and gerbil.

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