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Abstracts: CryoLetters 32 (3), 2011



Volume 32, No. 3 May/June 2011

ISSN 0143-2044



Cryopreservation of Garcinia cowa shoot tips by vitrification: the effects ofsucrose preculture and loading treatment on ultrastructural changes inmeristematic cells
Lip Vun Yap, Normah Mohd. Noor, Mahani Mansor Clyde and
Hoong Fong Chin




Cryopreservation of scalps of malaysian bananas using a pregrowth method
Philip Sipen, Paul Anthony and Michael R. Davey




High hatching rates after cryopreservation of hydrated cysts of the brine shrimp A. franciscana
Toru Yoshida, Yasuhiro Arii, Katsuhiko Hino, Ikuo Sawatani,
Midori Tanaka, Rei Takahashi, Toru Bando, Kazuhisa Mukai,
and Keisuke Fukuo




Thermodynamic aspects of cluster crystallization in cryoprotective solutions
A.I. Osetsky




Molecular cloning and characterization of two heat shock proteins in Thitarodes pui (lepidoptera: hepialidae)
Zhiwen Zou, Zixuan Sun, Junfeng Li and Guren Zhang




Effects of different cryoprotectants and cryopreservation protocols on the development of 2-4 cell mouse embryos
Wei-Xin LIU, Hua LU, Meng-Jun LUO, Liang-Zhi XU




Cryopreservation of Tetraclinis articulata (VAHL.) masters
Francisco Serrano-Martínez and José Luis Casas




Development of a cryopreservation procedure using aluminium cryo-plates
Shin-ichi Yamamoto, Tariq Rafique, Wickramage Saman
Priyantha, Kuniaki Fukui, Toshikazu Matsumoto
and Takao Niino




Cryopreservation of spleen and lymph nodes as a source of mononuclear cells to be used for the development of monoclonal antibody producing hybridoma cells
Ibrahim Sogut, Ibrahim Hatipoglu, Muge Serhatli,
Safak Isil Cevik, Deniz Durali and Aynur Basalp




Design of microprobe for accurate thermal treatment of tumor
Chao Chen, Aili Zhang, Zhanghao Cai, Jianqi Sun
and Lisa X. Xu





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CryoLetters 32 (3), 188-196 (2011)
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Lip Vun Yap1*, Normah Mohd. Noor2, Mahani Mansor Clyde1
and Hoong Fong Chin3

1Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D. E., Malaysia.
2Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D.E., Malaysia.
3Bioversity International, Regional Office for Asia, Pacific and Oceania, P.O. Box 236, UPM Post Office, 43400 Serdang, Selangor D. E. Malaysia.
*Corresponding author e-mail:


The effects of sucrose preculture duration and loading treatment on tolerance of Garcinia cowa shoot tips to cryopreservation using the PVS2 vitrification solution were investigated. Ultrastructural changes in meristematic cells at the end of the preculture and loading steps were followed in an attempt to understand the effects of these treatments on structural changes in cell membranes and organelles. Increasing preculture duration on 0.3 M sucrose medium from 0 to 3 days enhanced tolerance to PVS2 solution from 5.6% (no preculture) to 49.2% (3-day preculture). However, no survival was observed after cryopreservation. Examination of meristematic cells by transmission electron microscopy revealed the progressive accumulation of an electron-dense substance in line with increasing exposure durations to 0.3 M sucrose preculture. Treatment with a loading solution (2 M glycerol + 0.4 M sucrose) decreased tolerance of shoot tips to PVS2 vitrification solution and had a deleterious effect on the ultrastructure of G. cowa meristematic cells. This study suggests that G. cowa meristematic cells may lose their structural integrity due to exposure to glycerol present in the loading solution at a 2 M concentration, either due to its high osmotic potential, or due to its cytotoxicity.

Keywords: Garcinia cowa shoot tips; sucrose preculture; loading solution; dehydration tolerance; ultrastructure; transmission electron microscope (TEM).



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CryoLetters 32 (3), 197-205 (2011)
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Philip Sipen1, Paul Anthony2 and Michael R. Davey2*

1Department of Crop Science, Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia, Bintulu Campus, Nyabau Road, 97008 Bintulu, Sarawak, Malaysia.
2Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK.
*Corresponding author email:


The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 cm2) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light/8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40ºC for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg l-1 benzylaminopurine, 0.2 mg l-1 indole acetic acid and 10 mg l-1 ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48% of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10%, 13%, 42% and 48% for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.

Keywords: bananas, cryopreservation, meristems, Musa, pregrowth, scalps, sugars



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CryoLetters 32 (3), 206-215 (2011)
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Toru Yoshida1*, Yasuhiro Arii1, Katsuhiko Hino2, Ikuo Sawatani2,
Midori Tanaka1, Rei Takahashi3, Toru Bando4, Kazuhisa Mukai5
and Keisuke Fukuo1

1Department of Food Science and Nutrition, School of Human Environmental Sciences, Mukogawa Women's University, 6-46 Ikebiraki-cho, Nishinomiya, Hyogo, 663-8558 Japan.
2Research Center Glycoscience Institute, Hayashibara Biochemical Labs., Inc. 675-1 Fujisaki, Naka-ku, Okayama 702-8006 Japan.
3Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Doshisha Women's College of Liberal Arts, Kodo Kyotanabe, Kyoto 610-0395, Japan.
Department of Organ Preservation Technology, Kyoto University Hospital, 54 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
5Research & Development Center, Hayashibara Biochemical Labs., Inc. 1-2-3 Shimoishi, Kita-ku, Okayama, 700-0907 Japan.
*Corresponding author email:


Cysts of Artemia franciscana are known to be extremely tolerant to UV and ionizing radiation, hypoxia, dryness, osmotic pressure, and temperatures. However, when cysts are hydrated, their resistance to extreme environmental conditions is markedly reduced, and they subsequently enter a developmental sequence. The hatching rate of hydrated cysts declined when they were rapidly frozen after a short period of hydration but slow freezing improved hatching rates after 6-h hydration (1.4 g H2O /g dry wt). We observed that trehalose content in hydrated cysts was greatly reduced up to 6-h time. DSC analysis showed different thermal profiles at two cooling rates, suggesting the formation of a minuscule ice crystal inside the cells. High hatching rates can be obtained from highly hydrated cysts at slow cooling rate.

Keywords: cryopreservation; slow freezing; survival rate; trehalose.



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CryoLetters 32 (3), 216-224 (2011)
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A.I. Osetsky

Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, 23 Pereyaslavskaya str., 61015 Kharkov, Ukraine,


Crystallization of the solutions with quite a high intermolecular interaction of the components is analyzed. For the first time there has been considered the phenomenon of cluster crystallization of these solutions, enabling the reduction of total energy of intermolecular bonds, broken down during crystallization of the components has been discussed. A special priority is given to the cluster crystallization of aqueous solutions of cryoprotective substances close to vitrification temperature. Within this temperature range the mechanism of cluster crystallization is especially effective due to a sharp reduction of sizes of critical ice nucleation centers and diffusion mobility of molecules. This should be taken into account when designing the cryopreservation protocols for biological systems.

Keywords: cluster crystallization, critical nucleation center, cryoprotective solution, vitrification, state diagram.



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CryoLetters 32 (3), 225-239 (2011)
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Zhiwen Zou, Zixuan Sun, Junfeng Li and Guren Zhang*

State Key Laboratory for Biological Control, Sun Yat-sen University, Guangzhou, P. R. China 510275.
*Corresponding author e-mail:


As a group of proteins present almost in all organisms, heat shock proteins (HSPs) show transient expression in response to rapid temperature increase. The larvae of Thitarodes insects are the host of Ophiocordyceps sinensis, with high cold-tolerance. In order to study the adaptive mechanisms to temperature change, we cloned and sequenced the full-length cDNAs of two HSP genes (designated as tp-hsp90 and tp-hsp70) using the technique of rapid amplification of cDNA ends (RACE) from T. pui. The complete cDNA sequences of tp-hsp90 and tp-hsp70 are 2,842 bp and 2,169 bp long, encoding polypeptides of 712 and 651 amino acids with molecular weights of 81.57 and 71.27 kDa respectively. They show significant sequence similarity to homologous genes in insects. The inferred amino acid sequences of tp-hsp90 and tp-hsp70 are characterized by conserved features of HSP family: the proteins contain five signature sequences of HSP90 and three signatures of HSP70, respectively. Real-time quantitative reverse transcription-PCR (qRT-PCR) analyses show that tp-hsp90 expression is up-regulated in October and December, followed by a gradual rebound in January, March and May; while tp-hsp70 expression does not change significantly during the same period. These results suggest that tp-hsp90, rather than tp-hsp70, responds to temperature changes and should play a key role in cold tolerance in Thitarodes pui.

Keywords: HSP90, HSP70, cold tolerance, Thitarodes pui



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CryoLetters 32 (3), 240-247 (2011)
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Wei-Xin LIU1, 2, Hua LU 2, Meng-Jun LUO3 and Liang-Zhi XU1*

1West China Second Vniv Hospital, Sichuan University, Chengdu, 610041, China
2The Second Clinical College, Chengdu University of Chinese Traditional Medicine, Chengdu, 610041, China
3 Chengdu Institute of Family Planning, Chengdu, 610031, China
*Corresponding author e-mail:


This study was carried out to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of in vivo fertilised 2-4 cell mouse embryos. 2-4 cell mouse embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When 2-4 cell mouse embryos were cryopreserved by the slow-freezingôthe survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (P<0.05, respectively), but not significantly different among those of DMSO, G and EG (P>0.05, respectively), and not significantly different between those of PROH and EG (P>0.05, respectively). When 2-4 cell mouse embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (P<0.05, respectively). However, there were no significant differences among those of PROH, DMSO and G (P>0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of 2-4 cell mouse embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of 2-4 cell mouse embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.

Keywords: Mouse embryo; cryoprotectant; slow freezing; vitrification; embryo developmental potential



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CryoLetters 32 (3), 248-255 (2011)
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Francisco Serrano-Martínez and José Luis Casas*

Laboratorio de Biotecnología Vegetal. Instituto Universitario de Investigación CIBIO (Centro Iberoamericano de la Biodiversidad), Universidad de Alicante, Ctra. San Vicente del Raspeig s/n. E-03690 San Vicente del Raspeig, Alicante, Spain.
*Corresponding author email:


Tetraclinis articulata shoot tips excised from in vitro grown shoots were cryopreserved using a modified PVS2-based vitrification protocol. Preliminary experiments with non-cryostored shoot tips showed that the high concentrations of sucrose in loading (LS), vitrification (PVS2) and unloading (US) solutions employed in the protocol were very toxic for the explants. Replacement of sucrose by sorbitol in equal molar concentration in all these solutions enhanced survival of shoot tips after all treatments. However, cold-hardening of donor shoots before shoot tip excision was strictly required to obtain post-rewarming survival. Therefore, the protocol was outlined as follows: pre-conditioning of explants at 4ºC for 3 weeks in the dark; excision of 1 mm long shoot tips; loading for 20 min in modified LS at room temperature; dehydration in modified PVS2 at 0ºC for 60 min; immersion in liquid nitrogen (LN); rewarming at 40ºC for 2 min and subsequent transfer of shoot tips in modified US for 20 min.

Keywords: cryopreservation, cold acclimation, cryoprotectant solutions, Tetraclinis articulata, vitrification, conifers



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CryoLetters 32 (3), 256-265 (2011)
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Shin-ichi Yamamoto1, Tariq Rafique2, Wickramage Saman Priyantha3, Kuniaki Fukui1, Toshikazu Matsumoto4 and Takao Niino1*

1Genebank, National Institute of Agrobiological Sciences (NIAS), Kannondai 2-1-2, Tsukuba 305-8602, Japan.
2Plant Genetic Resource Programme (IABGR), National Agricultural Research Center, Islamabad, Pakistan.
3 Regional Agricultural Research and Development Center, Makandura, Gonawila, Sri Lanka.  
4Faculty of Life and Environmental Science, Shimane Univ., Matsue, Shimane 690-1102, Japan.
*Corresponding author email:


A cryopreservation procedure using an aluminium cryo-plate was successfully developed using in vitro-grown Dalmatian chrysanthemum (Tanacetum cinerariifolium) shoot tips. Shoot cultures were cold-hardened at 5°C on MS medium containing 0.5 M sucrose over a period of 20 to 40 days. Shoot tips with basal plate (1.0-1.5 x 1.0 mm) were dissected from shoot cultures and precultured at 5°C for 2 days on MS medium containing 0.5 M sucrose. Precultured shoot tips were placed on aluminium cryo-plates (7 mm x 37 mm x 0.5 mm) with 10 wells (diameter 1.5 mm, depth 0.75 mm) and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 or 60 min in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 1.4 M sucrose). For dehydration, the loading solution was replaced with PVS 7M vitrification solution (30% (w/v) glycerol, 19.5% (w/v) ethylene glycol and 0.6 M sucrose in liquid MS basal medium), which was applied for 40 min. After rapid immersion in liquid nitrogen, shoot tips attached to the cryo-plates were rewarmed by immersion in cryotubes containing 2 ml 1 M sucrose solution. Using this procedure, regrowth of cryopreserved shoot tips of line 28v-75 reached 77%. This protocol was successfully applied to six additional lines, with high regrowth percentages ranging from 65% to 90%. By contrast, the modified vitrification protocol tested as a reference produced only moderate regrowth percentages. This new method displays many advantages and will facilitate large scale cryostorage in genebank.

Abbreviations: BA, benzyladenine; LN, liquid nitrogen; MS, Murashige and Skoog; PVP, polyvinyl pyrolidone; PVS, plant vitrification solution; LS, loading solution.

Key words: aluminium plate, cryo-plate, Dalmatian chrysanthemum (Tanacetum cinerariifolium), vitrification, vitrification solution.



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CryoLetters 32 (3), 266-274 (2011)
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Ibrahim Sogut1, Ibrahim Hatipoglu1, Muge Serhatli1,
Safak Isil Cevik2,  Deniz Durali1 and Aynur Basalp1*


1TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, 41470 Gebze, Kocaeli, Turkey
2Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University. Orhanli, Tuzla, 34956 Istanbul,Turkey.
*Corresponding author e-mail:


In the present study, spleen and lymph nodes of mice were cryopreserved as a whole tissue and after thawing, membrane integrity of mononuclear cells was determined by trypan blue exclusion and PI staining. T and B lymphocytes, macrophages and dendritic cells have been isolated from both cryopreserved tissue and analyzed by Flow cytometry.  BALB/c mice were immunized with Hepatitis e antigen (HBeAg) and spleen and lymph nodes of mice were cryopreserved for 3 to 10 months. The cells obtained from both tissue were applied to hybridoma technology to understand if the cells keep their viability and functionality. The cells were isolated and fused with F0 mouse myeloma cells and several antibody producing hybrid cells were developed.  Results have shown that cryopreserved spleen and lymph nodes of mice can be efficiently used in hybridoma technology for the successful generation of monoclonal antibody producing hybrid cells.

Keywords: Cryopreservation, spleen, lymph nodes, hybridoma technology, ELISA, antibody



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CryoLetters 32 (3), 275-286 (2011)
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Chao Chen1, Aili Zhang1*, Zhanghao Cai2, Jianqi Sun2 and Lisa X. Xu1.2*

1Department of Biomedical Engineering, 2Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200240, P.R. China.
*Corresponding author e-mail:,


Thermal treatment has become an alternative modality for cancer treatment. Low temperature freezing and high temperature heating kill tumor cells effectively through direct and indirect injuries by biochemical and physical stresses. Hyperthermia at a mildly elevated temperature has also been reported to induce biochemical alternations to kill tumor cells and to stimulate immunological response to prevent metastasis. The comprehensive multi-scale biological responses to different thermal history experienced demand an accurate temperature control of the thermal system used for such a treatment.

A thermal system was built in our lab utilizing RF heating and liquid nitrogen cooling through a needle probe. In practice, difficulties involved in temperature measurement for in vivo monitoring and control of thermal input through two-phase LN2 flow inside the probe compromise the treatment outcome. To ensure an accurate temperature control, a new model was developed to study the dynamic freezing capacity of the cryo-probe by accounting for the probe shape and dimensions. The model was validated by experiments and used to predict the freezing processes under different conditions. Numerical simulation results showed that combined with RF heating, the system could be used to perform different treatment protocols with an accurate temperature control.

Keywords: Thermal treatment probe, two-phase flow, numerical simulation, accurate temperature control

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