Abstracts: CryoLetters 32 (2), 2011

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



Volume 32, No. 2 March/April 2011

ISSN 0143-2044



Effect of storage temperature on green spore longevity for the ferns Equisetum ramosissimum and Osmunda regalis
Daniel Ballesteros, Elena Estrelles, Christina Walters
and Ana M. Ibars




Cryopreservation of redwood (Sequoia sempervirens (D. Don.) Endl.) in vitro buds using vitrification-based techniques
E. A. Ozudogru, E. Kirdok, E. Kaya, M. Capuana, C. Benelli
and F. Engelmann




Limited elimination of two viruses by cryotherapy of pelargonium apices related to virus distribution
A. Gallard, R. Mallet, M. Chevalier and A.Grapin




Stability of lyophilized human platelets loaded with small molecule carbohydrates
JieXi Wang, Chao Yang, Yan Wang, MinXia Liu, SuPing Ren,
GuoBo Quan and Ying Han




Influence of low-molecular (below 5 kDa) fraction from cord blood and actovegin on phagocytic activity of frozen-thawed neutrophils
A.K. Gulevsky, N.N. Moiseyeva and O.L. Gorina




Gorgonian coral (Junceella juncea and Junceella fragilis) oocyte chilling sensitivity in the context of adenosine triphosphate response (ATP)
C. Lin, T. Zhang, F.W. Kuo and S. Tsai




Dimethyl sulfoxide and ethylene glycol promote membrane phase change during cryopreservation
Ralf Spindler, Willem F. Wolkers and Birgit Glasmacher




Cryopreservation of encapsulated liver spheroids using a cryogen-free cooler: high functional recovery using a multi-step cooling profile
Isobel Massie, Clare Selden, John Morris, Humphrey Hodgson
and Barry Fuller




Exploring the use of DMSO and Ascorbic acid to promote shoot development by excised embryonic axes of recalcitrant seeds
Cassandra Naidoo, Erica Benson, Patricia Berjak,
Meagan Goveia and N.W. Pammenter




Cryopreservation of apple in vitro axillary buds using droplet-vitrification
E. Condello, E.Caboni, E. Andrè, B. Piette, P. Druart,
R. Swennen and B. Panis




Book review - D.I. Berman, A.V. Alfimov, Z.A. Zhigulskaya
and A.N. Leirikh. 2010.
Overwintering and Cold-hardiness of Ants in the Northeast of Asia.
294 pp. Pensoft, Sofia-Moscow. ISBN: 978-954-642-549-2.





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CryoLetters 32 (2), 89-98 (2011)
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Daniel Ballesteros1,2*, Elena Estrelles1, Christina Walters2
and Ana M. Ibars1

1Banco de Germoplasma. Jardí Botànic de la Universitat de València-ICBiBE. C/Quart, 80. 46008. Valencia. Spain.
2USDA-ARS National Center for Genetic Resources Preservation, 1111 South Mason St. Fort Collins, CO, USA .
*Corresponding author


Green spores of ferns lose viability quickly, and need specialized treatment for long-term conservation in germplasm banks. Dry storage at different temperatures was studied in green spores of Osmunda regalis and Equisetum ramosissimum. Changes in germination percentage, time to 50% of maximum germination (T50) and tendency for normal growth of the gametophyte were assayed during 24 months of storage. Spores stored at 25ºC died within 1 month. Spores stored at 4ºC maintained high viability for about 3 months, and then aging was evident by a decrease of final germination percentage, an increase in T50, and abnormal development of the gametophyte. Germination of spores stored at -25ºC was highly variable during the storage period. Spores cryopreserved at -80ºC and -196ºC maintained high viability, rapid germination and normal growth throughout the study period. Cryopreservation of green spores is a feasible method to preserve viability and ensure normal gametophyte development for several years.

Keywords: cryopreservation, ferns, gametophyte development, green spore, spore germination, T50.



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CryoLetters 32 (2), 99-110 (2011)
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Cryopreservation of redwood (Sequoia sempervirens (D. Don.) Endl.) in vitro buds using vitrification-based techniques

E. A. Ozudogru 1*, E. Kirdok 1, E. Kaya 1, M. Capuana 2, C. Benelli 3
and F. Engelmann4, 5

1Gebze Institute of Technology, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul Caddesi, No 101, 41400, Gebze (Kocaeli), Turkey
2Istituto di Genetica Vegetale, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze), Italy.
3Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze), Italy.
4Institut de Recherche pour le Développement (IRD), UMR DIAPC, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France
5Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author email:


In this study, the efficiency of three vitrification-based cryopreservation techniques, i.e. vitrification, encapsulation-vitrification and droplet-vitrification were compared for cryopreserving Sequoia sempervirens apical and basal buds sampled from in vitro shoot cultures. The effect of cold-hardening of mother-plants and of bud culture medium and sucrose preculture was also investigated. Culture of apical and basal buds sampled from cold-hardened mother-plants on Quoirin and Lepoivre medium with activated charcoal had a positive effect on regrowth. Only droplet-vitrification ensured survival and regrowth after cryopreservation. After cryopreservation, regeneration of apical buds was possible for PVS2 exposure durations between 90 and 180 min but it remained low, with a maximum of 18% after 135 min treatment. With basal buds, regeneration after cryopreservation was possible over a larger range of PVS2 treatment durations, between 30 and 180 min. The highest regeneration percentage was slightly higher (22%) than that measured with apical buds, and was also achieved after 135 min PVS2 exposure.

Keywords: conservation, genetic resources, redwood, cryopreservation, droplet-vitrification.



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CryoLetters 32 (2), 111-122 (2011)
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A. Gallard1, R. Mallet2, M. Chevalier3 and A.Grapin1*

1AGROCAMPUS-OUEST, Centre d’Angers, INHP, UMR GenHort, 2 rue Le Nôtre 49045 Angers Cedex, France
2Service Commun d’Imageries et d’Analyses Microscopiques, Université d’Angers, France
3UMR GenHort, Centre INRA Angers-Nantes, Angers, France
*Corresponding author email:


The possibility of eradicating the pelargonium flower break virus (PFBV) and pelargonium line pattern virus (PLPV) by cryotherapy of axillary shoot apices was investigated using five Pelargonium cultivars. Viruses were detected by DAS-ELISA and their location was determined by immunolocalization. Apex culture did not permit elimination of PFBV and only 15% regenerated plants of ‘Stellar Artic’ cultivar were ELISA PLPV-negative. Plants regenerated from cryotherapy-treated apices were tested by DAS-ELISA after a 3-month in vitro culture period. Viruses were not detected in 25% and 50% of the plants tested for PFBV and PLPV, respectively. However, immunolocalization carried out on apices originating from cryopreserved shoot tips sampled from DAS-ELISA negative plants showed that they were still virus-infected. Using immunolocalization, PFBV and PLPV could be detected in Pelargonium apices, even in the meristematic dome. However, viral particles were more numerous in basal zone cells than in meristematic cells. Our results demonstrate that PFBV and PLPV are present within meristematic cells and that cryopreservation can partly reduce the quantity of these viruses in Pelargonium plants but not eliminate them totally. Additional knowledge on localization and behaviour of viruses during cryopreservation is essential to optimize cryotherapy and plant genetic resource management.

Keywords: Cryopreservation, droplet-vitrification, meristem culture, shoot tip, immunolocalization, PFBV, PLPV.



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CryoLetters 32 (2), 123-130 (2011)
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JieXi Wang, Chao Yang, Yan Wang, MinXia Liu, SuPing Ren,
GuoBo Quan and Ying Han*

Beijing Institute of Blood Transfusion, Academy of Military Medical Sciences, Beijing, China.
*Corresponding author: or


Long-term preservation of platelets is a great challenge for blood transfusion centers, due to the required narrow storage temperature arange (22 ± 2°C). Short shelf life and potential bacterial growth often lead to the shortage of high-quality platelets. Freeze-dried preservation is thus believed to be a potential solution for long-term platelet storage without losing the hemostasis function. Here we report a new platelet preservation method, which uses small molecule carbohydrates to extend storage time and to maintain platelet function. The activities of lyophilized platelets that were stabilized with small molecule carbohydrate (e.g., cell viability, mean platelet volume, activation characteristics, and aggregation kinetics) were maintained after storage of 30, 60, and 90 days at room temperature, 4°C, and -20°C. The recovery of freeze-dried platelets was 87% in comparison to fresh platelets. The mean platelet volume of rehydrated platelets increased (from 6.8 fl to 8.0 fl).  About 40% of rehydrated platelets was in the early-activated stage (PCA-1 positive) and 30% was in the terminal-activated stage (CD62P positive). The cell viability was about 60% as measured with CMFDA vital probes. The aggregation rate of rehydrated platelets after 90-day storage was similar to fresh platelets stored at 22°C ± 2°C.

Keywords: Platelets, Lyophilization, Preservation stability, Small molecule carbohydrate



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CryoLetters 32 (2), 131-140 (2011)
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Influence of low-molecular (below 5 kDa) fraction from cord blood and actovegin on phagocytic activity of frozen-thawed neutrophils

A.K. Gulevsky, N.N. Moiseyeva* and O.L. Gorina

Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine.
*Corresponding author: e-mail:


The influence of the cattle cord blood low-molecular fraction (below 5 kDa) as part of the rehabilitating medium in comparison with Actovegin on the functional activity of neutrophils after cryopreservation was studied. Incubation of frozen-thawed neutrophils in the rehabilitating media containing the low-molecular fraction or Actovegin was established to stimulate their phagocytic function, in particular engulfing and digesting ability. It was shown that after incubation of frozen-thawed neutrophils in the media containing CBF at the concentration 0.15 mg/ml or Actovegin at the concentration 1.5 mg/ml their oxygen-dependent metabolism was activated, since the number of NBT-positive neutrophils increased significantly in comparison with the control.  Gel-penetrating chromatography of CBF and Actovegin revealed differences between their chromatograms reflecting differences between the compositions compared. Our investigations proved that recovery of the functional activity of frozen-thawed neutrophils was possible in the media containing the cattle cord blood low-molecular fraction (below 5 kDa) or Actovegin at the concentrations 0.15 mg/ml and 1.5 mg/ml, respectively.

Keywords: cattle cord blood low-molecular fraction, Actovegin, frozen-thawed neutrophil, phagocytosis.



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CryoLetters 32 (2), 141-147 (2011)
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C. Lin1,2, T. Zhang4, F.W. Kuo1 and S. Tsai3*

1National Museum of Marine Biology & Aquarium, Checheng, Pingtung, Taiwan.
2Institute of Marine Biotechnology, National Dong Hwa University, Pingtung, Taiwan.
3Department of Biotechnology, Mingdao University, Peetow, Chang Hua, Taiwan.
4LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK.
*Corresponding author email:


Gorgonian corals are suffering continued decline in population and reproductive ability because of environmental changes. Cryopreservation can play an important role in ‘ex situ’ conservation for these corals. In the present study, oocyte chilling sensitivity in the context of adenosine triphosphate (ATP) response in two gorgonian species (Junceella juncea and Junceella fragilis) and the effectiveness of cryoprotectants in protecting coral oocytes from chilling injury were studied in an attempt to develop protocols for their cryopreservation. Oocytes of two gorgonian corals were exposed to methanol (1 M, 2 M) and EG (1 M) at 5, 0 and -5°C for up to 216 hours, and ATP levels in oocytes were then determined. ATP levels decreased gradually with exposure time and 1M methanol was more effective in protecting oocytes from chilling injury than other cryoprotectant treatments tested. J. juncea oocytes were less sensitive to chilling than J. fragilis oocytes. This study provided useful information for development of cryopreservtion protocols for the two gorgonian coral oocytes.

Keywords: gorgonian coral, oocyte, methanol, cryoprotectant, chilling sensitivity



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CryoLetters 32 (2), 148-157 (2011)
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Dimethyl sulfoxide and Ethylene Glycol promote membrane PHase change during cryopreservation

Ralf Spindler*, Willem F. Wolkers and Birgit Glasmacher

Institute for Multiphase Processes, Leibniz Universitaet Hannover, Germany.
*Corresponding author  e-mail:


Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to study the effects of dimethyl sulfoxide and ethylene glycol on cell pellets of human pulmonary microvascular endothelial cells during freezing from 4°C to –60°C at 1°C/min. FTIR analysis showed that membranes undergo a phase change in the presence of cryoprotective agents (CPAs) which was not observed in the absence of CPAs. Cryomicroscopy revealed the formation of intracellular ice and concomitant cell volume changes. Intracellular ice was detected in the majority of the cells both in the presence and absence of CPAs. Membrane phase changes were found to be most pronounced at intermediate concentrations of cryoprotective agents; for dimethyl sulfoxide at around 1 M and for ethylene glycol at around 1.5 M. At those concentrations cell survival after thawing exhibited a maximum. The results indicate that CPAs promote rather than prevent cell dehydration during freezing.

Keywords: FTIR, cryomicroscopy, dimethyl sulfoxide, ethylene glycol, dehydration, cryopreservation



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CryoLetters 32 (2),158-165 (2011)
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CRYOPRESERVATION OF ENCAPSULATED LIVER SPHEROIDS USING A CRYOGEN-FREE COOLER: high functional recovery using a multi-step cooling profile

Isobel Massie1*, Clare Selden1, John Morris2, Humphrey Hodgson1
and Barry Fuller1

1Royal Free Campus, UCL, Royal Free Hospital, Rowland Hill Street, London, NW3 2PF, UK
2Asymptote Ltd., St John's Innovation Centre, Cowley Road, Cambridge, CB4 0WS, UK.
*Corresponding author e-mail:


Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized.  A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12% v/v Me2SO/Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis.  The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to ~90%, 70% and 65% of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.

Keywords: cryopreservation, liver cell spheroids, alginate encapsulation, nitrogen, bioartificial liver, contamination



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CryoLetters 32 (2), 166-174 (2011)
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Cassandra Naidoo1, Erica Benson1,2, Patricia Berjak1,
Meagan Goveia1 and N.W. Pammenter1*

1University of KwaZulu-Natal, Durban, S. Africa;
2Damar Research Scientists, Drum Rd, Cuparmuir, Fife, Scotland, KY15 5RJ, UK
*Corresponding author contacts:


Seeds of Trichilia dregeana, T. emetica and Protorhus longifolia are recalcitrant (desiccation-sensitive), hence cryopreservation is the only ex situ means feasible for long-term conservation of these germplasm. For cryopreservation of these species, the excised embryonic axis is the explant of choice due to their small size and higher tolerance to desiccation. However, for many species with seeds having fleshy cotyledons, shoot development fails to occur after excision, which has been attributed to a reactive oxygen species (ROS) burst during excision wounding. This is a critical limiting step in developing cryopreservation protocols for such species. In embryos of T. dregeana, T. emetica and P. longifolia, the cotyledonary insertions are in close proximity to the shoot apical meristem and oxidative stress upon excision of the axis from cotyledons has been consistently associated with shoot tip necrosis, which precludes shoot development. This study tested the effects of dimethyl sulphoxide (DMSO) pre-culture prior to complete removal of the cotyledons, and post-excision soaking in DMSO or in the antioxidant, ascorbic acid, on shoot development by axes of T. dregeana and P. longifolia. These treatments had a significant (p<0.05) positive effect on shoot production with a 6 h DMSO pre-culture combined with a DMSO post-excision soak being optimal for promoting shoot production in 70% of the axes of T. dregeana and 60% of those of P. longifolia. Embryonic axes of T. emetica responded best to a 6 h DMSO pre-culture alone, with 55% of axes producing shoots. It was further shown that two different post-harvest developmental stages of T. dregeana axes differed significantly initially (p<0.05) in their response to DMSO and ascorbic acid treatments.

Keywords: antioxidants, ascorbic acid, cryopreservation, desiccation sensitive, DMSO, excision injury, Me2SO, oxidative stress, Protorhus, recalcitrant seeds, ROS, Trichilia



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CryoLetters 32 (2), 175-185 (2011)
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Cryopreservation of apple in vitro axillary buds using droplet-vitrification

E. Condello1, E.Caboni1*, E. Andrè2, B. Piette2, P. Druart3,
R. Swennen2 and B. Panis2

1CRA - Fruit Tree Research Institute, Via di Fioranello 52, 00134 Rome, Italy
2K.U. Leuven - Laboratory of Tropical Crop Improvement, Kasteelpark Arenberg 13, B-3001 Leuven, Belgium
3CRA W - Department Biotechnology, Chaussée de Charleroi 234, Gembloux, Belgium
*Corresponding author  e-mail:


In vitro axillary buds of two apple cultivars, Pinova and Jonagold, were successfully cryopreserved by droplet-vitrification. In vitro axillary buds of cv. Pinova were subjected to PVS2 for 15, 30, 45, 60, 80 or 100 min, while Jonagold buds were treated with PVS2 for 15, 30, 45 or 60 min. In addition, the effect of age of in vitro mother-plants on recovery after cryopreservation was evaluated. Recovery was performed on medium with various combinations of BA, IBA and GA3. Regrowth percentages for cv. Pinova increased in line with increasing PVS2 exposure durations, from 15 to 60 min. Cv. Jonagold showed a similar trend with an increase in regrowth from 30 to 60 min PVS2 exposure. Improved regrowth was observed when axillary buds were excised from aged mother-plants in comparison to those excised from plantlets that were regularly subcultured. The highest shoot regrowth was obtained when applying a 60 min PVS2 treatment to axillary buds excised from non-preconditioned 4-month old in vitro shoots and performing regrowth on recovery medium containing 4.50 M BA and 0.50 M IBA. This optimal protocol was also successfully applied to apple rootstocks M26 and
Jork 9.

Keywords: apple, axillary buds, cryopreservation, droplet-vitrification, growth regulators

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