CryoLetters

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

CryoLetters 22, 5-12 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Water relationships in Phyllantus orbicularis and Punica granatum antiviral extracts and Their influence on stability after FREEZING AND freeze-drying

J. Pendás¹, T. Moreira¹, O. Guerra², B. R. Peña³ and J.A Fernández³

¹ National Centre for Scientific Research (CNIC), PO Box 6990, Havana, Cuba
² Finlay Institute, PO Box 16017 Havana, Cuba
³ Faculty of Biology, University of Havana, Cuba

Summary

The effect of freezing, freeze-drying and storage on inhibitory half dose (ID₅₀) and on cytotoxic concentration (CC₅₀) of Phyllantus orbicularis and Punica granatum extracts was studied. Selective Index SI=(CC₅₀ /ID₅₀) was considered as the antiviral criterion. Both frozen and freeze-dried extracts were analyzed by differential scanning calorimetry (DSC). The values of vitreous transition temperatures of P. granatum (Tg’ and Tg) were lower than those of Ph. orbicularis. Sorption isotherms of freeze-dried products showed a typical sigmoidal shape, where GAB model was fitted instead of BET model. Water adsorption in freeze-dried Ph. orbicularis extract was higher than in the case of P.granatum. No significant changes in SI were induced by freezing or freeze-drying after a 6 months storage at -20ºC, but in the case of P.granatum extract, the effectiveness was highly affected after exposure to 37ºC or higher.

Keywords: Phyllantus orbicularis, Punica granatum, antiviral, freeze-drying, sorption isotherms, stability.

CryoLetters 22, 13-18 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

CRYOPRESERVATION OF AXILLARY BUDS OF A
 Eucalyptus grandis X Eucalyptus camaldulensis HYBRID

D Blakesley¹* and RJ Kiernan²

Department of Biology and Biochemistry, Faculty of Science, University of Bath, Claverton Down, Bath, BA2 7AY, UK
1.Department of Plant Breeding and Biotechnology, Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK (current address)
2.Shell Forestry, Horticulture Research International, East Malling, West Malling, Kent  ME19 6BJ, UK
*to whom correspondence should be addressed

Summary

This paper presents preliminary results from a study to develop methodology for the cryopreservation of axillary buds from an in vitro hybrid Eucalyptus grandis (W. Hill ex Maiden.) x Eucalyptus camaldulensis (Dehnnh.), maintained for use in a genetic modification programme. Axillary buds were encapsulated in an alginate gel, precultured on media containing elevated levels of sucrose, or a combination of sucrose and glycerol. Encapsulated buds were then dehydrated by evaporation prior to a two-step freezing process in liquid nitrogen. Eighteen percent of shoot explants survived freezing when sucrose alone was used as a protectant against dehydration and cryopreservation. Significantly higher survival (49%) was obtained with the incorporation of glycerol into the protocol. Following cryopreservation, shoots appeared to develop normally, with no evidence of adventitious meristems.

Keywords: Cryopreservation, Eucalyptus, sucrose, glycerol, conservation biotechnology

CryoLetters 22, 19-26 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

ULTRAPROFOUND CEREBRAL HYPOTHERMIA AND BLOOD SUBSTITUTION WITH AN ACELLULAR SYNTHETIC SOLUTION MAINTAINS NEURONAL VIABILITY IN RAT HIPPOCAMPUS

Milos Ikonomovic¹ , Kevin M. Kelly², Teresa M. Hentosz³, Shou-Ren Shih⁴,
David M. Armstrong⁵, Michael J. Taylor^4,6*

Department of Psychiatry¹, University of Pittsburgh Medical Center, Pittsburgh, PA; Departments of Neurology², Medicine³, and Surgery⁴, Allegheny General Hospital, Pittsburgh, PA; MCP Hahnemann University², Philadelphia, PA; Lankenaw Institute for Medical Research, Wynnewood, PA, and Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University⁵, Philadelphia, PA; Organ Recovery Systems, Inc., Suite 433, MSC 1119, Port City Center, 701 East Bay Street, Charleston, SC 29403, USA^4,6

Summary

The acute effects of ultraprofound hypothermia and blood substitution (UHBS) on neuronal cell viability were examined in adult rat hippocampus, a brain region particularly vulnerable to ischemic cell death. UHBS was performed using either artificial cerebrospinal fluid (ACSF) or Hypothermosol, an “intracellular-type” hypothermic preservation solution. After the procedure, the hippocampus was sliced and tested for cellular viability using a combination of cellular fluorochromes that are markers for live cells (acridine orange) and dead cells (propidium iodide). UHBS with ACSF resulted in a variable degree of neuronal death within the hippocampal subfields CA1/CA3, and dentate granular layer and hilus (CA4).  In contrast, UHBS with Hypothermosol consistently resulted in hippocampal slices with only mild neuronal death. Our results of preserved hippocampal neuronal viability with use of UHBS and Hypothermosol support the demonstrated central nervous system (CNS) protective effects of UHBS and Hypothermosol when used during prolonged cardiac arrest. The results of this study also suggest that UHBS and Hypothermosol may be useful in the preparation and maintenance of viable hippocampal tissue for physiological studies, especially those involving aged animals, which are particularly vulnerable to hypoxic-ischemic cellular injury.

Keywords: Aging, hippocampus, hypothermia, viability, ischemia, hypoxia

CryoLetters 22, 27-34 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

CRYOPRESERVATION OF AURICULA SHOOT TIPS USING THE ENCAPSULATION/DEHYDRATION TECHNIQUE

R. Hornung^1*, A. Holland¹, H.F. Taylor¹ & P.T. Lynch²

¹ Horticulture Section, Wye College, Wye, Ashford, Kent, TN25 5AH, UK.
² Division of Biological Sciences, School of Environmental and Applied Sciences, University of Derby, Kedleston Road, Derby DE22 1GB, UK.
To whom correspondence should be addressed (e.mail: shs-rh@wye.ac.uk).

Summary

Calcium alginate encapsulated, in vitro culture derived shoot tips of the florist's auricula, cultivar Embley, were successfully cryopreserved using the encapsulation/dehydration technique. The use of preculture media containing both sucrose and mannitol was essential for post-thaw growth. After 2 weeks cold acclimatisation at 10C, shoot tips were encapsulated and incubated in culture medium containing 0.25 M sucrose and 0.25 M mannitol for 48h, followed by 23h dehydration  with silica gel (water content of beads approximately 16%) prior to plunging into liquid nitrogen. Post-thaw growth of the shoot tips was without callus formation. The morphology of frozen and non-frozen shoots was comparable.

Keywords: Auricula, Cryopreservation, Encapsulation/dehydration, Sugar preculture

CryoLetters 22, 35-42 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

THE SEQUENCE OF THE PREPARATIVE PROCEDURES
AFFECTS THE SUCCESS OF CRYOSTORAGE OF CASSAVA SOMATIC EMBRYOS

Philippa Stewart,  Michelle Taylor and David Mycock*

Department of Animal, Plant & Environmental Sciences, University of the Witwatersrand, Private Bag X3, Wits, 2050, Johannesburg, South Africa

Summary

Cassava primary somatic embryos were prepared for cryostorage using both physical drying and cryoprotectant application stages. The effects of the sequence by which these preparative events were applied to the material was investigated. The drying of the somatic embryos before the application of the cryoprotectants resulted in a significantly higher viability after cryostorage than when the material was first exposed to the cryoprotectants and then dried. The impact of these preparative procedures on sub-cellular detail was monitored by means of transmission electron microscopy.

Keywords: cassava, drying, cryoprotectant, ultrastructure

CryoLetters 22, 43-50 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

CRYOPRESERVATION OF Taxus chinensis SUSPENSION CELL CULTURES

Sang-Ic Kim, Hyung-Kyoon Choi, Joo-Sun Son, Jeong-Hwan Yun, Moon-Suk Jang,
Hong-Rak Kim, Jai-Young Song, Jin-Hyun Kim, Ho-Joon Choi and Seung-Suh Hong*

Samyang Genex Biotech Research Institute,63-2, Hwaam-Dong, Yusung-Gu, Taejon, 305-348, Korea
* To whom correspondence should be addressed (E-mail: sshong@genex.co.kr)

Summary

A simple cryopreservation method for suspension cells of Taxus chinensis was established. In this procedure 7 days old suspension cells were used without any pre-culture treatment. At first, cells were incubated in cryoprotectant solution (0.5M DMSO and 0.5M glycerol) on ice for 30 min and then frozen at a cooling rate of 1ºC/min to -40ºC prior to immersion in liquid nitrogen. The average viability of frozen-thawed cells was between 30 to 40%. The recovery of cryopreserved cells in liquid nitrogen for 1 month was accomplished. After rapid thawing, cells were transferred to solid medium and cultivated for 4-6 weeks. The treatment of trehalose as a cryoprotectant enhanced re-growth of frozen-thawed cells. The stable maintenance of paclitaxel biosynthetic ability in cryopreserved cells was confirmed by comparing with that of regularly sub-cultured suspension cells.

Keywords: Taxus chinensis, suspension cell culture, cryopreservation, paclitaxel, Trehalose

CryoLetters 22, 51-60 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

Phenomenological Theory on Refrigeration effect of practical type- Superconductor

L. Shan, D.W. Lu, S.A. Aruna*, N.L. Chen, X.N. Xu, X. Jin

Department of Physics and National Laboratory of Solid State Microstructure, Nanjing University, Nanjing 210008, China
*Department of Physics, Fourah Bay College, University of Sierra Leone, Freetown, Sierra Leone

Summary

In accordance with the phenomenological theory of thermodynamics, we discussed the Magnetocaloric effect of type- superconductors, which are exposed to a varying magnetic field. It is concluded theoretically that the refrigeration effect is closely associated with demagnetization effect for practical specimens, which is different from the situation for ideal superconductor sample having no demagnetization effect. From the thermodynamic formulation of superconductivity, we deduced the integrating function of the thermal effect. By numerical calculation, we found a quantitative relation between the refrigeration effect and the demagnetizing factor which is determined by the shape of the sample.

Keywords: Refrigeration; Type- superconductor; Demagnetizing factor

CryoLetters 22, 61-74 (2001)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU

routine cryopreservation of kiwifruit (Actinidia spp) germplasm by encapsulation–dehydration:
Importance of plant growth regulators

Y. Bachiri¹, G.Q. Song², P. Plessis¹, A. Shoar–Ghaffari¹, T. Rekab¹ and C. Morisset¹*

¹ Laboratoire de Cryobiologie Végétale - Université Pierre et Marie CURIE, 12, Rue Cuvier,  75252 PARIS, France – E-mail: <christiane.morisset@wanadoo.fr>
² Plant Physiology and Biochemistry Laboratory – China Agricultural University 100094,  BEIJING, CHINA

Summary

The encapsulation-dehydration protocol was optimized for an in vitro cultured hybrid Actinidia arguta  A. deliciosa. Shoot tips from 14-d reactivated mononodal microcuttings were embedded, transferred to liquid culture medium whose sucrose concentration was daily increased (0.3, 0.5, 0.75 M) and then kept at 0.75 M for 2 or 4 d. Dehydration on silica gel was monitored to 20±1.5% residual water content (dry weight basis), allowing direct quenching in liquid nitrogen and rewarming at room temperature. Differential scanning calorimetry analysis underlined the importance of reversible glass transition in shoots for survival. Regrowth ranged from 85% to 95%. Growing shoot tips showed no phenotypic abnormalities. Rooting was also achieved. This method was routinely applied to diploid A. chinensis and A. eriantha, and to several diploid hybrids, yielding over 70% regrowth. A slight decrease in sucrose molarity (0.65 M) allowed tetraploid A. chinensis and A. chrysantha x A. arguta to survive dehydration, but not quenching in LN. For A. deliciosa cv Hayward and cv Tomuri, normal regrowth after cryopreservation was achieved only after modification of the pre- and post-culture media, highlighting the importance of monitoring plant growth regulator balance, principally at the post-thaw recovery step.

Keywords:  Actinidia, Cryopreservation, dehydration, differential scanning calorimetry (DSC), encapsulation, sucrose.

Abstracts

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