Abstracts: CryoLetters 29 (2), 2008

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

CryoLetters 29(2), 89-94 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

High viability of dormant malus buds after 10 years of storage in liquid nitrogen vapour

G.M. Volk1*, J. Waddell1, R. Bonnart1, L. Towill1, D. Ellis1 and M. Luffman2

1USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA
2Canadian Clonal Genebank, Agriculture and Agri-Food Canada, 2585 County Road 20, Harrow, ON N0R 1G0
*Correspondence author:  G M Volk    e-mail:


Three hundred and sixty two Malus accessions from the Canadian Clonal Genebank of Plant Gene Resources of Canada were cryopreserved as dormant buds at the USDA-ARS National Center for Genetic Resources Preservation in 1996.  According to grafting data collected on 165 of these accessions in 1999, 80% of the accessions had at least 40% viability. A subsample of these accessions was processed for cryopreservation by either adjusting the moisture content of the budwood sections containing dormant buds to 32 or 37% moisture (fresh weight basis) or by not drying the budwood sections (46% moisture fresh weight basis) prior to cooling.  Budwood sections were then slow-cooled at 1ºC h-1 to -30ºC, held for 24 h at -30ºC and then rapidly transferred to the vapour phase of liquid nitrogen.  Cryopreserved buds from 13 accessions that were dried using the various techniques were warmed and grafted in both 1999 and 2006 to determine viability. Overall, bud viability was high at both storage times.  At the 10 year timepoint, some accessions had higher bud growth when they were desiccated prior to slow-cooling when compared to those that were not. 

Keywords: apple, cryopreservation, genetic resources, genebank



CryoLetters 29(2), 95-110 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jayanthi Nadarajan1*2a, Marzalina Mansor2,  Baskaran Krishnapillay2, Harry J. Staines1, Erica E. Benson3 and Keith Harding3

1University of Abertay Dundee, Bell Street. Dundee DD1 1HG, Scotland, UK
2Forest Research Institute of Malaysia (FRIM), Kepong, 52109 Kuala Lumpur, Malaysia
3Research Scientists, Conservation, Environmental Science and Biotechnology, Damar, Drum Road, Cupar Muir, Fife, KY15 5RJ, Scotland, UK
*Author for correspondence (email:
a(Current contact address) Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK


Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential Scanning Calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.21 ± 1.07ºC) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.22 ± 0.95ºC as the sugar’s concentration was decreased to 2.5% (w/v). Tg heat capacity for shoot-tips treated with 2.5% and 5% (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 ± 0.05 to 0.23 ± 0.01 J.g-1 respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was >150 J.g-1 and decreased to ca. 60 J.g-1 with 2.5% (w/v) trehalose.  For 5% and 10% (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J.g-1 respectively and freezing points were depressed to –75ºC and –85ºC with 2.5% and 5% trehalose (w/v), respectively.  DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70% survival was found optimal for stable glass formation during cooling and on rewarming.

Keywords: Parkia speciosa, differential scanning calorimetry, vitrification, trehalose 



CryoLetters 29(2), 111-119 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Yan-Ping Wang1, Xue-Ming Zhao1, Guang-Bin Zhou1,3, Yun-Peng Hou1,2,Zhi-Qiang Fan1, Chang-Liang Yan1, Lun-Suo1, Xiang-Wei Fu1 and Shi-En Zhu1,2*

1 Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing 100094, P.R. China.
2 State Key Laboratories for Agrobiotechnology, China Agricultural University, Beijing 100094, P.R. China.
3 Dujiangyan campus of Sichuan Agricultural University, Dujiangyan 611830, P.R. China


The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized withfrozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0~60%) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2%) and blastocysts formation rates (63.6%) were obtained when the egg yolk concentration was adjusted to 30%. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40°C before plunging into liquid nitrogen (LN2). After thawing, the highest cleavage rate (87.4%) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2%, 65.4%) and Group 3 (72.5%, 45.0%) were higher (P < 0.05) than those in Group 2 (22.2% and 13.9%), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1% and 22.7% of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.

Keywords: mice, spermatozoa, oocyte, cryopreservation, ZIP, in vitro fertilization



CryoLetters 29(2), 121-133 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


María Eugenia Mamprin1*, Silvana Petrocelli1, Edgardo Guibert2 and Joaquín Rodríguez1

1Farmacología and Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002 LRK Rosario, Argentina.
2Farmacologia, Depto Cs. Fisiologicas, Facultad de Cs. Bioquimicas y Farmaceuticas, UNR, Suipacha 531, S2002LRK Rosario, Argentina.


We have used hepatocyte suspensions to study how hypothermic storage in a modified University of Wisconsin (mUW) solution affects liver cell metabolism and cell membrane properties. At present the UW solution is the gold standard of organ preservation. However it contains several ingredients which either are expensive or cannot easily be obtained worldwide. The aim of the present study was the development of a novel preservation solution for rat hepatocytes effective and comparable with the mUW, with enhanced buffer capacity and less expensive. In particular, we investigated the effects of the buffering agent BES, a derivate of aminosulfonic acid, on liver cells metabolism during cold storage and rewarming. In the development of this novel preservation solution we have included three key components: gluconate as impermeant anion, sucrose to give additional osmotic support and the aminosulfonic acid BES for its buffer capacity. Our results shown that BGS solution was equally effective to mUW to protect rat hepatocytes against cold preservation injury due to ischemia and reoxigenation. Also, BGS solution is a good alternative with its high buffer capacity, best indices of respiration activity and it is considerably less expensive than the mUW solution. The use of this solution for the storage of isolated hepatocytes may facilitate hepatocyte research in situations in which the more complex and expensive mUW solution is not available since the cost of one liter of BGS is about 1/3 that an equal volume of mUW solution.

Keywords: hepatocytes, BGS solution, cold storage



CryoLetters 29(2), 135-144 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

DEVELOPING cryopreservation FOR picea sitchensis (sitka spruce) somatic embryos: A compariSON OF vitrification protocols

Samantha Gale1,2,  Allan John2,  Keith Harding1,3 and Erica E Benson1,3*

1Plant Conservation Group, Univ of Abertay Dundee, Bell Street, Dundee, DD1 1HG, UK.
2Forestry Commission, Northern Research Station, Roslin, Midlothian, UK.
3Current/Contact Address: Research Scientists, Conservation, Environmental Science & Biotechnology, Damar, Drum Road, Cuparmuir, Fife, KY15 5RJ, UK.
*Corresponding author: Erica Benson, e-mail:


Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, ~10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P<0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation/dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.

Keywords: clonal forestry, somatic embryos, vitrification, cryobanking



CryoLetters 29(2), 145-156 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum L.) shoot tips

A. Kaczmarczyk*, T. Rutten, M. Melzer and E.R.J. Keller

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany.


The ultrastructure of cells within shoot tips of S. tuberosum ‘Désirée’ was studied after different steps of the DMSO droplet cryopreservation method. After 2 h of DMSO treatment, cells contained numerous small vesicles, while at the same time mitochondria and chloroplasts had increased in size and vacuoles had assumed an irregular shape. After rapid cooling in liquid nitrogen, subsequent rewarming, and 1 h incubation there were no apparent changes in the ultrastructural organization of the cells, suggesting that they might be still intact. However, two days after rewarming, the meristematic dome area and part of the epidermis showed signs of extensive damage. Rupture of plasmalemma, plasmolysis and destruction of cell organelles as well as strong heterochromatisation of nuclei were observed. Survival and regeneration of cells were found mainly in leaf primordial regions. Here cells were very active, containing many mitochondria and intact or regenerating chloroplasts. Alternating temperature preculture of donor plants before shoot tip isolation improved the cryopreservation results (plant regeneration 46.5 %) as compared to constantly warm precultured shoot tips (plant regeneration 20.0 %), which showed slightly stronger damage after rewarming from liquid nitrogen.

Keywords: Solanum tuberosum ‘Désirée’, cell damage, cryo injury, DMSO droplet method



CryoLetters 29(2), 157-164 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Peter Whittaker

Centre for Economic and Social Aspects of Genomics, Institute for Advanced Studies,
County South, Lancaster University, Lancaster LA1 4YD, UK.


An account of the sources and properties of human stem cells is given with an indication of ethical issues associated with each.  The ethical considerations of umbilical cord blood stem cell banks are considered in more detail. It is suggested that some private stem cell banks may not, at present, be providing an ethically valid service.  Public stem cell banks, on the other hand, may be more likely to save lives although it will be necessary to assure their long-term financial status.

Keywords: stem cells, stem cell banks, ethics.



CryoLetters 29(2), 165-174 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Mathew Tomlinson

Fertility unit, Nottingham University Hospitals (Queens Campus), Derby Rd, Nottingham NG7 2UH


The process of cryopreservation of cells and tissues either for research or for therapeutic use is loaded with risk from beginning to end. Centres must focus on key areas of potential hazard or incident, particularly those associated with injury, loss or damage to stored material, and misidentification of stored material. Incidents involving any would have more fundamental consequences for the laboratory including financial loss and a threat to continuation of a service or project. Control measures to prevent injury are as much about education in the use and handling of liquid nitrogen as they are about protective clothing and more attention should be focused on preventing explosion, asphyxiation, burns and injury from manual handling. Other major losses are focused on damage to samples due to inappropriate processing, inadvertent thaw or less likely, contamination from another sample or microorganism. Complete sample losses can result from misidentification, poor inventory control and poor record keeping with regard to the use of samples in therapeutic procedures. Control and prevention of these hazards may require significant resources, although more often than not involve simple changes in procedure, ensuring that equipment and materials are fit for purpose and that personnel are suitably trained. Identification of such risk relies on a combination of a systematic prospective approach (risk assessment) with a documented and formal recognition of previous mistakes or near misses. Risk potential can be formally scored and ranked to provide services with the means of prioritising and allocating appropriate resources. Failure to implement a comprehensive risk management strategy could be translated as ‘negligence’ should a similar incident subsequently occur. The risk management of any services including cryopreservation should therefore be considered fundamental and integrated into business planning, objective setting and departmental budgeting to ensure continued improvement and the delivery of a quality and safe service or indeed research project.

Keywords: Risk assessment, cryopreservation, sperm storage, embryo storage



CryoLetters 29(2), 175-179 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Low Temperature organ preservation, blood vessels and the human tissue Act in 2007: IMPACT AND IMPLICATIONS

Barry J Fuller1,2* and Sas Dijk1

1University Department of Surgery, Royal Free & UCL Medical School, Hampstead Campus, UCL
2Royal Free Hospital, London NW3 2QG, UK
*Corresponding author: Barry Fuller, e-mail:


The impact of the enactment of the Human Tissue Act is discussed in relation to procedures currently applied during cadaveric organ procurement for transplantation within the UK. The major effect has been to regulate the storage of blood vessels for use as accessory grafts during transplantation, where hypothermic preservation beyond 2 days has historically been practiced and now falls within the remit of the Act concerning storage of human tissues. The benefits and drawbacks of considering cryopreserved blood vessels in such situations are also debated.    

Keywords: organ preservation; blood vessels; hypothermic storage, the Human Tissue Act.

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