Abstracts: CryoLetters 28 (3), 2007

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

CryoLetters 28 (3), 137-150 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Duško P. Blagojević

Institute for Biological Research, Department for Physiology, Bulevar despota Stefana 142, 11060 Belgrade, Serbia.


Hetero and endothermic adaptive responses arising as a result of natural responses  to environmental cues include antioxidant systems that support adaptations to environmental low temperatures in the broadest sense. These temperatures induce phase changes in energy production and consequently changes in the concentration of reactive oxygen species (ROS). The latter may lead to oxidative stress and the impairment of cellular homeostasis and antioxidant defence systems (ADS) scavenge the ROS so generated. In endotherms the ADS responds to oxidative pressure during acute cold stress conditions, this response is tissue specific and does not extend to prevent other oxidative damage. The early acute phase of cold exposure is accompanied by a significant depletion in redox equivalents. Under such conditions it is questionable if ADS has the capacity to neutralize elevated levels of ROS since there is also an increased energy demand and enhanced ATP consumption. Prolonged exposure to cold leads to ADS adaptation. Hibernators and freeze-tolerant species elevate their ADS before hibernation or freezing in order to prepare for and cope with re-awakening. The involvement of ROS and the role of the ADS in organisms subjected to low tempreatures are features intercalated into physiological mechanisms of homestasis. The exact mechanisms for ADS regulation have not been fully defined and are the subject of many ongoing intruiging scientific investigations.

Keywords: Free radicals, reactive oxygen species, antioxidants, low-temperature biology



CryoLetters 28 (3), 151-172 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Vitrification, encapsulation-VITRIFICATION AND droplet-vitrification: a review

Akira Sakai1 and Florent Engelmann2, 3*

1 Hokkaido University (retired), Asabucho 1-5-23, Kitaku, Sapporo, 001 Japan.
2 Institut de recherche pour le développement (IRD), UMR 1097, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France.
3 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


 This paper discusses the importance of the successive steps of the vitrification technique and reviews the current development and use of vitrification and of the two derived protocols, encapsulation-vitrification and droplet-vitrification. Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Samples are thus cryopreserved without detrimental intracellular ice formation. In a standard vitrification protocol, excised explants are precultured on medium enriched with sucrose, treated (‘loaded’) with a loading solution composed of 2 M glycerol + 0.4 M sucrose, dehydrated with a highly concentrated vitrification solution [e.g. the PVS2 vitrification solution, which contains 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) DMSO and 0.4 M sucrose], frozen and rewarmed rapidly, unloaded with basal culture medium supplemented with 1.2 M sucrose, and then transferred to standard culture conditions. In the encapsulation-vitrification technique, the explants are encapsulated in alginate beads, loaded and dehydrated with a vitrification solution before rapid immersion in liquid nitrogen. In the droplet-freezing technique, excised explants are loaded, treated with the vitrification solution and frozen in individual microdroplets of vitrification solution placed on aluminium foils, which are immersed rapidly in liquid nitrogen. These three techniques have been applied to different tissues of over 100 plant species from temperate and tropical origins and the number of cases where they are being tested on a large scale or applied routinely is increasing.

Keywords: plant germplasm; long-term conservation; cryopreservation; vitrification; encapsulation-vitrification; droplet-vitrification.



CryoLetters 28 (3), 173-186 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A Parametric Study of Freezing Injury in BPH1CAFTD-2 Human Prostate Tumor Cells

Matthew G. Geeslin3, David J. Swanlund1 and John C. Bischof*1,2,3

Departments of Mechanical Engineering1, Urologic Surgery2, and Biomedical Engineering3, University of Minnesota, Minneapolis, Minnesota.
*1100 Mechanical Engineering, 111 Church St. S.E., Minneapolis, Minnesota 55455.


Freeze injury in BPH1CAFTD-2 cells frozen/thawed in suspension was studied through a two-level four-parameter (24) experimental design and analysis.  The four parameters considered were end temperature, hold time, TNFα concentration, and thawing rate.  Thermal parameter values chosen were based on the approximate thermal history cells would experience in the peripheral region of a cryosurgical iceball. Cell suspensions were frozen at a constant 10°C/min on a directional solidification stage and viability was assessed within 1 hr post-thaw using a dye exclusion assay.  The parameters affecting cell survival were determined through calculation of the individual parameter effects (E) and interactions (I) according to factorial design guidelines; data set curvature (C) was also determined.  Cell viability ranged from a maximum of 87.55% to a minimum of 17.59% indicating trends in cell survival were sensitive to the parameters chosen.  Survival was affected by the following parameters in order: lowering the end temperature, increasing the hold time, adding TNFα, and reducing the thawing rate. In addition, all 2-way parameter interactions except TNFα-hold time were statistically significant. Curvature analysis showed that cell viability at the midpoint of the data was nearly 20% lower than predicted based on linear interpolation. These results were verified and extended using analysis of variance (ANOVA).  We conclude that cryoinjury in this tumor line can be influenced by multiple interacting thermal parameters, most importantly end-temperature and hold time, as well as the presence of the cytokine TNFα. Finally, although the cell type is tumorigenic results suggest the possibility of using freezing to control benign prostatic hyperplasia (BPH) in addition to cancer within the prostate.

Keywords: prostate cancer, BPH1CAFTD-2 cells, freezing, TNFα, parametric design, ANOVA



CryoLetters 28 (3), 187-196 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

FREEZE-DRYING OF HUMAN PLATELETS: influence of saccharide, freezing rate and cell concentration

Xin-Li Zhou1, Hong Zhu3, Shao-Zhi Zhang2, Fa-Ming Zhu3, Guang-Ming Chen2*, Li-Xing Yan3

1Institute of Biothermal Sciences, University of Shanghai for Science and Technology, Shanghai 200093, China.
2Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou 310027, China.
3Blood Center of Zhejiang Province, Hangzhou 310006, China.


Freeze-drying of human platelets is one potentially ideal approach for long-term preservation of platelets. In this study, effects of concentration and type of saccharides, freezing rate and initial cell concentration on the recovery of freeze-dried platelets were investigated. Annexin V binding platelet activation assays, scanning electron microscopy and platelet aggregation upon thrombin (1 U/ml) addition were used to evaluate the effectiveness of platelet freeze-drying. The numerical recovery of freeze-dried platelets was reached as high as (93.0±5.2) % and the recovery of nonactived platelets was reached up to (85.7±3.4) % in the presence of 1%BSA and 20% trehalose. Frozen by shelf pre-cooling was the best way to freeze the sample in this study and the numerical recovery of freeze-dried platelets was reached (93.0±5.2) % at about 10 ºC/min. When the platelet concentration was increased from 0.2 to 4×109 platelets/ml, recovery remained higher than 81.4%. The morphology of freeze-dried and rehydrated platelets was intact but a little rounder compared with fresh platelets. The maximum aggregation rate to thrombin (1 U/ml) of freeze-dried platelets was 83.9% of the fresh ones, but aggregation speed was 43.0% of the fresh ones. Further research on rehydration process and scale up are required.

Keywords: Platelets; Freeze-drying; Lyoprotectant; Cooling rate; Cell concentration



CryoLetters 28 (3), 197-206 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Takao Niino1*, Daisuke Tanaka1, Raminosoa Rivo Tantely2,
Kuniaki Fukui1and Kazuto Shirata1

1 Genebank, National Institute of Agrobiological Sciences (NIAS), Kannondai 2-1-2, Tsukuba 305-8602, Japan,
2 Centre National de Recherches sur l’Environnement, Antananarivo 101, Madagascar


An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 µM BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25°C. Clumps that grew many buds were cold-hardened at 5°C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5°C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25°C. Then they were dehydrated in 1 ml PVS2 solution at 25°C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa ‘NZ219’ buds reached 87%. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30% to 90% and an average of 63%.

Keywords: Mat rush, igusa, cryopreservation, vitrification, multiple stemmed culture



CryoLetters 28 (3), 207-213 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Mi Jin Seo1, Jong Hee Shin2 and Jae Keun Sohn1*

1Department of Agronomy, Kyungpook National University, Daegu, Republic of Korea,
2Institute for Bioresources Research, Gyeongbuk Provincial Agricultural Technology Administration, Andong, Republic of Korea.


A simplified technique for the cryoprotection of dormant shoot-tips was developed for the germplasm conservation of herbaceous peony (Paeonia lactiflora Pall.). The highest post-thaw regrowth level was obtained from shoot-tips desiccated by air drying for 5 h. The post-thaw regrowth of P. lactiflora. var. 'Mikang' was the highest (74.9%) among the five genotypes tested. The regeneration level after cryopreservation was highest (66~74%; average of 70%) for dormant shoot-tips collected from November to February. Shoot tips which were dissected from non-dormant buds in late March, however, showed very low post-thaw regrowth. Post-thaw regrowth of shoot-tips was promoted in MS medium containing 1 mg l-1 of gibberellic acid (GA3) and 0.5 mg l-1 of N6-benzyladenine (BA). The elongated shoots were rooted on a 1/4 MS medium containing 0.1 mg l-1 of β-naphthalene acetic acid (NAA). In addition, random amplified polymorphic DNAs (RAPDs) assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the peony dormant shoot-tips. This cryopreservation method appears to be promising for the conservation of herbaceous peony germplasm.

Keywords: cryopreservation, dormant bud, desiccation, herbaceous peony.



CryoLetters 28 (3), 217-222 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Quan Sheng Zhang1,2, Yi Zhou Cong3, Shan Cun Qu3,
Shi Ju Luo 3 and Xue Xi Tang1,*

1 Ecology Laboratory, Ocean University of China, Qingdao, P.R. China
2 Ocean School, Yantai University, Yantai, P.R. China
3 Shandong Oriental Ocean Sci-tech Co., Ltd., Yantai, P.R. China
* Corresponding author.  E-mail:


Little attention has been given to the effect of interactions between different cryogenic parameters on the viability of cryopreserved algae. Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen by two-step cooling, and interactions were tested for optimizing the cooling protocol and improving freeze-tolerance. UNOVA of a general linear model suggested that interactions between both cooling rate and holding time and between holding temperature and holding time significantly affected the survival of thawed gametophytes. Based on the magnitude of effect, the importance order of factors was found to be: holding temperature, holding time, cooling rate, cooling rate × holding temperature, holding temperature × holding time. UNOVA also suggested significant main effects of pre-culture conditions and sex on survival of thawed gametophytes. Under the optimal procedure, post-thaw survivals obtained were 84% for male and 69% for female gametophytes. Male gametophytes were observed to be more tolerant to the whole procedure of cryopreservation than females. Following thawing viable gametophytes could grow asexually or give rise to morphologically normal sporophytes.

Keywords: Cryopreservation, Laminaria japonica, gametophytes, macroalgae, two-step cooling, interaction

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