Abstracts: CryoLetters 31 (4), 2010

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



Volume 31, No. 4 July/August 2010

ISSN 0143-2044



The effect of tween 80 on eggshell permeabilization in galleria mellonella (L.) (lepidoptera, pyralidae)
Elena Cosi, Muhamad T. Abidalla and Pio Federico Roversi




Comparison of vitrification and encapsulation-dehydration for cryopreservation of Thymus moroderi shoot tips
Ana Marco-Medina, José Luis Casas and M. Elena González-Benito




Comparing cooling systems for the cobe 2991 cell separator used in the purification of human pancreatic islets of langerhans
YA. Adewola, R. Mage, M. Hansen, B. Barbaro, J. Mendoza-Elias,
T. Harvat, PH. Morel, J. Oberholze, and Y.Wang




In vitro 17ß-oestradiol release as a marker for follicular survival in cryopreserved intact bovine ovaries
R. Gerritse, R. Peek, F.C.G.J. Sweep, C.M.G. Thomas, D.D.M. Braat,
J.A.M. Kremer, J.R. Westphal and C.C.M. Beerendonk




Do ice nucleating agents limit the supercooling ability of the land snail cornu aspersum?
Armelle Ansart, Annegret Nicolai, Philippe Vernon and Luc Madec




Direct cryopreservation of winter buds of nine cultivars of blackcurrant (Ribes nigrum L.)
Jon Green and Brian Grout




Vitrification-based cryopreservation of Grammatophyllum speciosum protocorms
Kathawut Sopalun, Kanchit Thammasiri and Keiko Ishikawa




Meeting announcements; Society for Low Temperature Biology, Society for Cryobiology; Erratum – CryoLetters 31 No 3, pp 200-205





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CryoLetters 31 (4), 291-300 (2010)
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the effect of Tween 80 On eggshell permeabilizATION IN galleria mellonella (L.) (lepidoptera, pyralidae)

Elena Cosi1, Muhamad T. Abidalla2 and Pio Federico Roversi1*

1Agriculture Research Council (CRA), Research Center for Agrobiology and Pedology, Cascine del Riccio, Via di Lanciola 12/A, 50125 Florence, Italy.
2Biology and Biotechnology Department, University of Basilicata, Potenza 85100, Italy
*Corresponding author  e-mail:


The development of a species-specific protocol for dechorionation/permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2% in the early stage and from 4.3 to 11.2% in the late stage. The embryos treated with 1.25% NaOCl + 0.08% Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 ± 1.5%) and late stages (22.4 ± 3.7%). Thus, 0.08% Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Keywords: Galleria mellonella, eggshell, dechorionation, permeabilization, Tween 80, cryopreservation.



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CryoLetters 31 (4), 301-309 (2010)
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Ana Marco-Medina1, José Luis Casas1* and M. Elena González-Benito2

1Laboratorio de Biotecnología Vegetal. Instituto 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)
2 Departamento de Biología Vegetal, E.U.I.T. Agrícola, Ciudad Universitaria, E-28040 Madrid (Spain)
*Corresponding author  e-mail:


Vitrification and encapsulation-dehydration were tested for cryopreservation of Thymus moroderi Pau ex Martínez (Labiatae), an endemic plant from south-eastern Spain. For vitrification, shoot tips were loaded in a solution containing 0.4 M sucrose + 2 M glycerol for 20 min at room temperature, dehydrated in PVS2 solution for 0-105 min at 0ºC, then immersed in liquid nitrogen (LN) for at least 1 day and rapidly rewarmed. The highest survival (71.4%) was obtained after 60 min PVS2 dehydration. Encapsulation-dehydration gave slightly lower results, with up to 50% explants survival. In the optimal protocol, donor plants were cold-hardened at 10ºC for 5 weeks, excised shoot tips precultured for 48 h on MS medium with 0.08 M sucrose, encapsulated, pretreated in medium with 0.75 M sucrose for 19 h, desiccated to 22% moisture content (fresh weight basis), and immersed in LN. Vitrification thus appears more suitable than encapsulation-dehydration for cryopreservation of T. moroderi shoot tips.

Keywords: cryopreservation, encapsulation-dehydration, Thymus sp., vitrification



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CryoLetters 31 (4), 310-317 (2010)
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A. Adewola1, R. Mage2, M. Hansen1, B. Barbaro1, J. Mendoza-Elias1, T. Harvat1, PH. Morel2, J. Oberholzer1*, and Y.Wang1

1Department of Transplant/Surgery, University of Illinois at Chicago;
2Clinic for Digestive and Transplant Surgery, Department of Surgery, University Hospital, Geneva, Switzerland.
Y. Wang, University of Illinois at Chicago, Department of Transplant/Surgery, Clinical Science Building, Suite 402, Chicago, Illinois 60612.
*Corresponding author  email:


Two different approaches of controlled cooling of the Cobe 2991 cell-separator for islet purification were evaluated. The first method is the new Geneva COBE cooling system (GCCS), which consists of an electronically controlled liquid nitrogen injection system. The second is the University of Illinois at Chicago cooling system (UICCS), which consists of a specially designed “Cold Room” maintained at 1-8°C. For the GCCS, the mean temperatures of the gradient solutions were measured at the beginning and end of centrifugation were found to be 7°±0.7C and 6.8±0.6°C respectively. For the UICCS, the mean temperature of the gradients at the beginning and end of centrifugation were 4.7°C±0.53°C and 7.03°C±0.91°C respectively. The presented COBE cooling systems can easily be adapted to a COBE 2991 cell-separator and are efficient in maintaining gradient solutions at a defined low temperature during centrifugation.

Keywords: COBE cell-separator, temperature, islet, purification, gradients, cooling



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CryoLetters 31 (4), 318-328 (2010)
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R. Gerritse1, R. Peek2*, F.C.G.J. Sweep3, C.M.G. Thomas3, D.D.M. Braat2, J.A.M. Kremer2, J.R. Westphal2 and C.C.M. Beerendonk2

1Streekziekenhuis Koningin Beatrix, Department of Obstetrics and Gynecology, Winterswijk, The Netherlands; Radboud University Nijmegen Medical Centre,
2Department of Obstetrics and Gynecology
3Department of Chemical Endocrinology, Nijmegen, The Netherlands.
*Corresponding author  e-mail:


Transplantation of cryopreserved intact ovaries from cancer patients is a technically challenging option for restoring fertility after sterilizing cancer therapy. In this paper we describe an assay based on 17ß-oestradiol (oestradiol) production, to monitor the functional damage sustained by the ovarian tissue during the freeze/thawing procedure. To this end, fresh bovine ovarian cortical biopsies were cultured in vitro for 7 days. As a control, the oestradiol release of biopsies that had sustained maximal cryodamage was analyzed. In addition the oestradiol release by cortical biopsies from two ME2SO perfused and cryopreserved intact ovaries was analyzed. Oestradiol production could be measured in culture supernatants, while oestradiol release of maximal cryo-damaged biopsies was at background levels. In vitro oestradiol release by cortical biopsies can be used as a functional marker for cryo-damage and indicates that our assay is suitable to optimize the cryopreservation procedure of intact ovaries.

Keywords: ovary, cryopreservation, oestradiol, in vitro assay, intact organ



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CryoLetters 31 (4), 329-340 (2010)
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Do ice nucleating agents limit the supercooling ability of the land snail Cornu aspersum?

Armelle Ansart1*, Annegret Nicolai1, Philippe Vernon2 and Luc Madec1

1 Université de Rennes 1, UMR 6553 Ecobio, bâtiment 14A, 263 Avenue du Général Leclerc, CS 74205, 35042 Rennes Cedex, France.
2 Université de Rennes 1, UMR 6553 Ecobio, Station Biologique, 35380 Paimpont, France
*Corresponding author  e-mail :


The supercooling ability of adults and eggs of the partially freezing tolerant land snail Cornu aspersum remains limited to high subzero temperatures (ca. -5°C) whatever the conditions, suggesting the presence of ice nucleating agents (INAs). In this study, we investigated the nucleation activity of the digestive tract of adult snails, eggs and their direct environment: food, faeces and soil. The mucous ribbon always present in the distal intestine of adults exhibited a heat-sensitive (i.e. organic) nucleation activity, close to that of the entire snails during dormant states (aestivation and hibernation). However, a microbial nature of these INAs could not be established in inactive snails. The food provided to active snails contained ice nucleating bacteria, which followed the digestive tract to be found in the intestine and in the faeces, but with a decreasing concentration along the transit. Eggshells also presented a heat-sensitive nucleation activity, which could be related to its structure. Moreover, eggs are laid directly in the soil which contained both organic and mineral INAs. This study is the first to demonstrate the implication of organic INAs in the cold hardiness of a terrestrial gastropod.

Keywords: cold hardiness, ice nucleating agents, ice nucleating bacteria, gastropod



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CryoLetters 31 (4), 347-357 (2010)
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Kathawut Sopalun1, Kanchit Thammasiri2,3* and Keiko Ishikawa4

1Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Phyathai, Bangkok 10400, Thailand.
2Department of Plant Science, Faculty of Science, Mahidol University, Rama VI Road, Phyathai, Bangkok 10400, Thailand.
3Institute of Molecular Biosciences, Mahidol University, Putthamonthon, Nakornpratom 73170, Thailand.
4Department of Research and Development, Japan Horticultural Production and Research Institute, Kamishiki, Matsudo, Chiba, 270-2221, Japan.
*Correspondence author   e-mail:


Three vitrification-based methods for the cryopreservation of Grammatophyllum speciosum protocorms were invesigated: droplet-vitrification, encapsulation-dehydration and encapsulation-vitrification. Protocorms, 0.1 cm in diameter, developed from 2-month-old germinating seeds were used. For droplet-vitrification, protocorms were precultured on filter paper soaked in half strength Murashige and Skoog medium (½MS) containing 0.4 M sucrose at 25  2ºC for 2 d, followed by soaking in loading solution (2 M glycerol and 0.4 M sucrose in ½MS liquid medium) for 20 min and then dehydrated with PVS2 solution [30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in ½MS liquid medium containing 0.4 M sucrose at pH 5.7] for 30 min. For encapsulation-dehydration, encapsulated protocorms were precultured in ½MS liquid medium containing 0.4 M sucrose on a shaker (110 rpm) at 25  2ºC for 2 d, followed by soaking in the same loading solution for 20 min and then exposed to a sterile air-flow at 2.5 inches/water column from the laminar air-flow cabinet for 8 h. For encapsulation-vitrification, encapsulated protocorms were precultured in ½MS liquid medium containing 0.4 M sucrose for 1 or 2 d, followed by soaking in the same loading solution for 20 min and then dehydrated with PVS2 solution for 60 min. For all three methods, preculturing with 0.4 M sucrose for 2 d resulted in a significant induction of dehydration and freezing tolerance. The cryopreservation results showed highest protocorm regrowth after droplet-vitrification (38%), followed by encapsulation-dehydration (24%) and encapsulation-vitrification (14%). Plantlets developed from these three methods did not show any abnormal characteristics or ploidy level change when investigated by flow cytometry.

Keywords: orchid, cryopreservation, protocorms, droplet-vitrification, encapsulation-dehydration, encapsulation-vitrification.

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