Abstracts: CryoLetters 33 (1), 2012

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



Volume 33, No. 1 January/February 2012

ISSN 0143-2044



Gene expression profiling of a cold-shocked earthworm Eisenia andrei
Hyeng-Soo Kim, Chi Hyun Ahn, Yong-Soo Park,
Hum Dai Park, Ki Seok Koh, Zae Young Ryoo,
Soon Cheol Park, and Sanggyu Lee




V-cryo-plate procedure as an effective protocol for cryobanks: case study of Mint cryopreservation
Shin-ichi Yamamoto, Tariq Rafique, Kuniaki Fukui,
Kentaro Sekizawa and Takao Niino




Effect of cytochalasin b pre-treatment of in vitro matured porcine oocytes before vitrification
F. Marco-Jiménez, L. Casares-Crespo and J.S. Vicente




Assessment of external heat transfer coefficient during oocyte vitrification in liquid and slush nitrogen using numerical simulations to determine cooling rates
M.V. Santos, M. Sansinena, N. Zaritzky and J. Chirife




Effect of “ice blockers” in solutions for vitrification of in vitro matured ovine oocytes
Francisco Marco-Jimenez, Fiammetta Berlinguer,
Giovanni G. Leoni, Sara Succu and Salvatore Naitana




Cryobanking of Korean Allium germplasm collections: results from a 10 year experience
Haeng-Hoon Kim, Elena Popova, Dong-Jin Shin,
Jung-Yoon Yi, Chun Hwan Kim, Jae-Sun Lee,
Moo-Kyoung Yoon and Florent Engelmann




Vitrification-based cryopreservation of shoot-tips of Pinus kesiya Royle ex. Gord.
Varginia Kalita, Hiranjit Choudhury*, Suman Kumaria
and Pramod Tandon




Cryopreservation of in vitro grown shoot tips of Diospyros kaki Thumb. using different methods
Niu Yanli, Luo Zhengrong, Zhang Yanfang and Zhang Qinglin




Expression and distribution of aquaporin 8 in rat hepatocytes cold stored 72 hours in modified university of wisconsin and bes-gluconate-sucrose solutions. Study of their correlation with water content
Miszczuk G, Mediavilla MG, Pizarro MD, Tiribelli C,
Rodríguez J and Mamprin ME




Editorial Announcements and Upcoming Meetings





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CryoLetters 33 (1), 1-11 (2012)
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Hyeng-Soo Kim1, Chi Hyun Ahn2, Yong-Soo Park3, Hum Dai Park4, Ki Seok Koh5, Zae Young Ryoo1, Soon Cheol Park2, and Sanggyu Lee1*

1School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea
2Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea
3Kyongbuk Livestock Research Institute, 66-1 Mt., Mookri, Anjung, Youngju, Kyongbuk 750-871, Republic of Korea
4Department of Biotechnology, School of Engineering, Daegu University, 15 Jillyang, Gyeongsang, Gyeongbuk, 712-714, Republic of Korea
5Department of Anatomy, Konkuk University College of Medicine, Seoul 143-701, Republic of Korea
*Corresponding author  e-mail:


To identify genes that are modulated under cold-stress conditions in the earthworm Eisenia andrei, we performed a genome-wide analysis of gene expression in cold-shocked earthworms by using Serial Analysis of Gene Expression (SAGE). We identified 5,977 and 5,407 unique SAGE tags under normal and cold-stressed conditions, respectively. The majority of the SAGE tags did not match to any known expressed sequences, due to a paucity of expression data in earthworms. We converted the statistically significant SAGE tags for the cold-stressed condition into expressed sequence tags (ESTs), and the results showed that particular genes associated with energy homeostasis, cellular defense mechanisms, and ion balance were up-regulated or down-regulated. We constructed a regulatory network of some of these genes and identified rps-6 as a core gene in the cold-response regulatory-gene network. Our data provide a baseline for gene expression studies of cold shock in the Lumbricidae.

Keywords: earthworm, Eisenia andrei, cold stress, SAGE, transcription, gene expression



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CryoLetters 33 (1), 12-23 (2012)
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Shin-ichi Yamamoto1, Tariq Rafique2, Kuniaki Fukui1, Kentaro Sekizawa3 and Takao Niino1*

1Genebank, National Institute of Agrobiological Sciences (NIAS), Kannondai 2-1-2, Tsukuba 305-8602, Japan,
2Plant Genetic Resource Programme, Institute of Agri-Biotechnology and Genetic Resources, National Agricultural Research Center, Islamabad, Pakistan
3National Center for Seeds and Seedlings, Tsukuba 305-0852, Japan
*Corresponding author  e-mail:



A vitrification procedure using aluminium cryo-plates (V-Cryo-plate procedure) was successfully developed and adjusted for in vitro-grown mint (Mentha spp.) shoot tips. Shoots were cultured at 25°C on MS medium containing 0.088 M sucrose for 7 to 14 days after the last subculture. Shoot tips with a basal part (1-1.5 mm × 1 mm) were dissected from the shoots and precultured at 25°C for 1 day on the same medium. Precultured shoot tips were placed on aluminium cryo-plates with 10 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25°C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in 25 ml pipetting reservoirs filled with PVS2 for 20 min at 25°C. Then the cryo-plates were transferred in uncapped 2 ml cryotubes and directly plunged into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in cryotubes containing 2 ml 1 M sucrose solution at room temperature. Using this procedure, regrowth of cryopreserved shoot tips of line ‘Fukuyamajisei’ reached over 90%. This protocol was successfully applied to 16 additional Mentha lines, with regrowth ranging from 73% to 100%. This V-Cryo-plate method will facilitate the cryostorage of mint germplasm in our genebank.

Keywords: aluminium plate, cryo-plate, mint (Mentha spp.), vitrification, V-Cryo-plate method

Abbreviations: LN, liquid nitrogen; LS, loading solution; MS, Murashige and Skoog; PVS, plant vitrification solution



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CryoLetters 33 (1), 24-30 (2012)
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Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification

F. Marco-Jiménez*, L. Casares-Crespo and J.S. Vicente

Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain


The study tested the efficacy of pre-treating mature porcine oocytes with cytochalasin B before vitrification by the open pulled straw method (OPS) in a low toxicity solution containing ice blockers. The effects of pre-treating the oocytes with 7.5 μg/ml cytochalasin B before vitrification on membrane integrity, chromosome organisation and cortical granule distribution were evaluated. When oocytes pre-treated with cytochalasin B before vitrification were compared with control oocytes, similar membrane integrity was observed. In contrast, when both vitrified oocytes groups (treated and untreated with cytochalasin B) were compared with fresh oocytes, significantly lower proportions of oocytes with normal chromosomes aligned regularly on the metaphase plate and peripheral cortical granule distribution were observed.  The percentages of oocytes with normal chromosomes aligned regularly on the metaphase plate were similar between those treated or untreated with cytochalasin B before vitrification. Similar results were found for normal cortical granules distribution.  Irrespective of previous cytochalasin B exposure, vitrification gave rise to higher abnormal cortical granule distribution percentages.  Cytochalasin B pretreatment of oocytes before vitrification does not help to reduce the damage induced by the cryopreservation process of porcine oocytes.

Keywords: cytoskeleton; OPS; Ice blockers; VM3



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CryoLetters 33 (1), 31-40 (2012)
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Assessment of external heat transfer coefficient during oocyte vitrification in liquid and slush nitrogen using numerical simulations to determine cooling rates

M.V. Santos1, M. Sansinena2*, N. Zaritzky2 and J. Chirife1

1Depto. de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional de La Plata and Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CONICET-UNLP) , La Plata, Argentina
2Facultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, Buenos Aires, Argentina.
*Corresponding author  email:


In oocyte vitrification, plunging directly into liquid nitrogen favor film boiling and strong nitrogen vaporization. A survey of literature values of heat transfer coefficients (h) for film boiling of small metal objects with different geometries plunged in liquid nitrogen revealed values between 125 to 1000 W/(m2 K). These h values were used in a numerical simulation of cooling rates of two oocyte vitrification devices (open-pulled straw and Cryotop®), plunged in liquid and slush nitrogen conditions. Heat conduction equation with convective boundary condition was considered a linear mathematical problem and was solved using the finite element method applying the variational formulation. COMSOL Multiphysics was used to simulate the cooling process of the systems. Predicted cooling rates for OPS and Cryotop when cooled at -196ºC (liquid nitrogen) or -207ºC (average for slush nitrogen) for heat transfer coefficients estimated to be representative of film boiling, indicated lowering the cooling temperature produces only a maximum 10% increase in cooling rates; confirming the main benefit of plunging in slush over liquid nitrogen does not arise from their temperature difference. Numerical simulations also demonstrated that an hypothetical four-fold increase in the cooling rate of vitrification devices when plunging in slush nitrogen would be explained by an increase in heat transfer coefficient. This improvement in heat transfer (i.e., high cooling rates) in slush nitrogen is attributed to less or null film boiling when a sample is placed in slush (mixture of liquid and solid nitrogen) because it first melts the solid nitrogen before causing the liquid to boil and form a film.

Key words: vitrification, oocyte, heat transfer coefficient, cooling rate, slush nitrogen, finite element analysis.



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CryoLetters 33 (1), 41-44 (2012)
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Effect of “Ice Blockers” in solutions for vitrification of in vitro matured ovine oocytes

Francisco Marco-Jimenez1*, Fiammetta Berlinguer2, Giovanni G. Leoni3, Sara Succu2 and Salvatore Naitana2

1Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Spain. 46022
2Department of Animal Biology, University of Sassari, Sassari, Italy
3Department of Physiological, Biochemical and Cellular Science, University of Sassari, Sassari, Italy
*Corresponding author  email:


Polymers have been used as a substitute for serum in vitrification solutions for embryos and oocytes. This study was designed to replace serum with defined commercial macromolecules in vitrification solution for in vitro matured ovine oocytes.  Oocytes were cryopreserved in two vitrification solutions (16.5% ethylene glycol + 16.5% dimethyl sulphoxide) supplemented with 1% of SuperCool X-1000 and 1% SuperCool Z-1000 (Ice Blockers) or 20% foetal calf serum (FCS). After warming, oocytes viability and developmental potential after processing for in vitro embryo production were assessed.  The number of viable oocytes (87.4% and 85.9%), cleaveage rates (21.4% and 19.6%) and blastocyst development rates (4.8% and 4.5%) were similar for Ice Blockers and FCS, respectively. On the basis of these findings, it may be concluded that combined use of Ice Blockers (SuperCool X-1000 and SuperCool Z-1000) as supplementation in vitrification solution offers similar results to serum for vitrification of in vitro matured ovine oocytes.

Keywords: SuperCool X-1000; SuperCool Z-1000; Vitrification; Cryotop; Oocyte; Ovine



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CryoLetters 33 (1), 45-57 (2012)
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Haeng-Hoon Kim1,2*, Elena Popova1,3, Dong-Jin Shin4, Jung-Yoon Yi1, Chun Hwan Kim5,    Jae-Sun Lee6, Moo-Kyoung Yoon7 and Florent Engelmann8,9

1National Agrobiodiversity Center, RDA-NAAS, Suwon 441-707, Korea
2Bioversity International, Regional Office for Asia, Pacific and Oceania, P.O. Box 236, UPM Post Office, 43400 Serdang, Selangor D. E. Malaysia. (present address)
3Korea Forest Research Institute, Suwon 441-847, Korea (present address)
4Species Restoration Center, Korea National Park Service, Gurye 542-853, Korea
5Agricultural Research Center for Climate Change, NIHHS, RDA, Jeju 690-150, Korea
6Danyang Garlic Experiment Station, Danyang 395-841, Korea
7National Institute of Horticultural & Herbal Science, RDA, Suwon, 440-706, Korea
8Institut de recherche pour le développement (IRD), UMR DIADE, BP 64501, 34394 Montpellier cedex 5, France
9Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy
*Corresponding author email:


This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9% after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.

Keywords: cryopreservation, cryotherapy, droplet-vitrification, garlic, implementation.



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CryoLetters 33 (1), 58-68 (2012)
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vitrification-based cryopreservation of shoot-TIPS of Pinus kesiya royle ex. gord.

Varginia Kalita1, Hiranjit Choudhury*2, Suman Kumaria1 and Pramod Tandon1

1Division of Plant Biotechnology, Department of Botany, School of Life Sciences, North-Eastern Hill University, Shillong 793022, Meghalaya, India
2Biotechnology Division, Department of Basic Sciences & Social Sciences, School of Technology, North-Eastern Hill University, Shillong 793022, Meghalaya, India
*Corresponding author e-mail:


The present investigation was aimed at developing a protocol for long-term preservation of germplasm of Pinus kesiya Royle ex. Gord. through vitrification. Some of the critical components affecting explant tolerance to cryopreservation, such as effects of preculture, vitrification solutions, exposure time to vitrification solutions, volume of vitrification solution and its toxicity, washing of vitrified tissues after thawing, were analysed. The results showed that shoot regrowth of P. kesiya shoot-tips was considerably affected when exposed to cryoprotectants for longer periods of time (> 10 min). Among different vitrification solutions studied, maximum survival (76%) of shoot-tips was achieved with mVSL (using 0.6 ml of the solution) in MS basal medium containing 4.0 mg l-1 N6-benzyladenine (BA).

Keywords: regrowth, thawing, germplasm, LN, Pinus kesiya, BA- N6-benzyladenine.



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CryoLetters 33 (1), 68-74 (2012)
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Niu Yanli 1, 2, Luo Zhengrong 1*, Zhang Yanfang 1 and Zhang Qinglin1

1Key Laboratory of Horticultural Plant Biology Affiliated to Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China;
2Chinese Academy of Science, Lushan Botanical Garden, Lushan, Jiangxi, 332900, China
*Corresponding author  email:


The objective of this study was to compare the potential of different cryopreservation strategies for in vitro shoot tips of Diospyros kaki Thunb. The treatments consisted of three different cryopreservation methods: vitrification, droplet-vitrification and modified droplet-vitrification. The following variables were assessed: cold acclimation, sucrose concentration in the preculture medium and PVS2 treatment time. A higher average survival level was obtained using the modified droplet-vitrification method compared to the other two methods.

Keywords: Diospyros kaki Thunb., vitrification, droplet-vitrification, modified droplet -vitrification



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CryoLetters 33 (1), 75-81 (2012)
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Miszczuk G1, Mediavilla MG1, Pizarro MD1, Tiribelli C2, Rodríguez J1 and Mamprin ME1,3,*

1Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC) Universidad Nacional de Rosario Avda. Arijón 28 bis, Rosario (S2011BXN) Argentina
2Centro Studi Fegato, AREA Science Park, Basovizza SS 14, Km 163,5. 34012 Trieste and Department of Medical Sciences, University of Trieste, Italy
3Farmacologia, Depto Cs. Fisiologicas, Facultad de Cs. Bioquimicas y Farmaceuticas, UNR. Reprints requests to María E Mamprin, CAIC, Avda. Arijón 28 bis, Rosario (S2011BXN), Argentina.
*Corresponding author  email:


Since few data are availble on the genetic responses to low temperatures, we investigated if cold storage of hepatocytes (0°C, mUW or BGS solutions, 72 h) can affect gene expression and/or cellular localization of AQP8 and their correlation with water movements. Cold preserved hepatocytes showed a significant decrease in water content (P<0.05) but were able to regulate their volume when they returned to physiological conditions. These changes were not related to modulation in the expression and the pattern of distribution of AQP8 suggesting that other mechanisms are involved. The study of the quantitative changes in the expression of genes coding for liver specific proteins in cold preserved hepatic cells is of interest in order to develop new preservation methods or solutions that could contribute to maintain the utility of these cells when destined to be applied in clinical models.

Keywords: hepatocytes, aquaporin 8, cold storage, BGS solution

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