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Abstracts: CryoLetters 28 (5), 2007

CryoLetters 28 (5), 313-328 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

COLD PRESERVATION OF ISOLATED HEPATOCYTES IN UW SOLUTION. EXPERIMENTAL STUDIES ON THE RESPIRATORY ACTIVITY AT 0şC

Maria Soledad Llarrull1, Maria Dolores Pizarro1, Angel L. Scandizzi1, Hebe Bottai2, Edgardo E. Guibert3 and Joaquin V. Rodriguez*

1Farmacología, Departamento de Ciencias Fisiológicas;
2Estadística, Departamento de Ciencias Matemáticas;
3Biología Molecular, Departamento de Ciencias Biológicas; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531-S2002LRK Rosario, Argentina.
*Address correspondence and reprints requests to Joaquin V. Rodriguez, Farmacologia, Depto Cs. Fisiologicas, Facultad de Cs. Bioquimicas y Farmaceuticas, UNR, Suipacha 531, S2002LRK Rosario, Argentina. Email:
jrodrig@fbioyf.unr.edu.ar

Abstract

To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0°C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0°C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0şC was determined using a modified physical method and it was 410 µM O2, which, as expected, is lower than the solubility in water at the same temperature (453 µM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 ± 0.13 nmol/O2/min/106 cells, which represents 1% of the control value at 36 °C (61.46 ± 14.61 nmol/O2/min/106 cells). The respiratory activity and cell viability were well maintained during the preservation period.
At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.

Keywords: oxygen, hepatocytes, cell respiration, UW, cold preservation

 

 

CryoLetters 28 (5), 329-336 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

  • REGENERATION OF DIOSCOREA FLORIBUNDA PLANTS FROM CRYOPRESERVED ENCAPSULATED SHOOT TIPS: EFFECT OF PLANT GROWTH REGULATORS
  • B.B. Mandal* and Sangeeta Ahuja-Ghosh1
  • Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India.
    1701 S. Zarzamora, Texas Diabetes Institute, San Antonio, Texas 78207-5209, USA (current address).
    * To whom correspondence should be addressed:
    mandalbinay@yahoo.co.in

    Abstract

    The encapsulation-dehydration protocol for the cryopreservation of in vitro shoot tips of Dioscorea floribunda was optimized. Maximum survival of 87% was obtained when overnight pretreatment with 0.3 M sucrose was followed by encapsulation, preculture in 0.75 M sucrose for 4 d, dehydration in a laminar air flow for 5˝ h, quenching in liquid nitrogen and thawing at 40oC. During recovery growth, 29% shoot formation was obtained when cryopreserved shoot tips were initially cultured for 25 d on a medium with 1.5 mg l-1 BAP, 0.2 mg l-1 NAA and 0.2 mg l-1 GA3 followed by culturing for 15 d on a medium with reduced  BAP (1 mg l-1) but increased NAA (0.5 mg l-1) and GA3 (0.3 mg l-1). Finally, transfer on to a medium with further reduced doses of BAP (0.05 mg l-1) and NAA (0.15mg l-1) but without GA3 stimulated production of fully grown plantlets. All plants regenerated without callus formation. Modification of post-thaw culture media with plant growth regulators was essential for regrowth of shoot tips to plantlets.

    Key words: cryopreservation, encapsulation-dehydration, plant growth regulators.

    Abbreviations: BAP: 6-benzylaminopurine; NAA: naphthaleneacetic acid; GA3: gibberellic acid; LN: liquid nitrogen; MS: Murashige & Skoog's medium; RM: regrowth medium; PGR: plant growth regulator.

     

     

    CryoLetters 28 (5), 337-346 (2007)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

    INVESTIGATION OF THE INFLUENCE OF CELL DENSITY OF HUMAN FIBROBLASTS CRYOPRESERVED INSIDE COLLAGEN SPONGES AT VARIOUS COOLING RATES

    Yumi Matsumura1, Masanobu Ujihira1,2*, Satoshi Nogawa2, Keita Kimura2, Hinako Ichikawa2 and Kiyoshi Mabuchi1,2

    1Graduate School of Medical Sciences and
    2School of Allied Health Sciences, Kitasato University  1-15-1 Kitazato Sagamihara-shi, Kanagawa 228-8555, Japan
    *Email:
    uj@kitasato-u.ac.jp.

    Abstract

    The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 105 to 107 cells/cm3. Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50°C/min in a solution of Dulbecco's Modified Eagle Medium, 20% Fetal Bovine Serum and 10% dimethylsulfoxide, kept frozen below –185°C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.

    Keywords: Artificial Tissue, Cell Density, Collagen Matrix, Cryopreservation, Human Fibroblast, Post-Thaw Membrane Integrity of Cells

     

     

    CryoLetters 28 (5), 347-358 (2007)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

    COLD TOLERANCE AND OVERWINTERING OF AN INTRODUCED NEW ZEALAND FROG, THE BROWN TREE FROG (LITORIA EWINGII)

    Y. Bazin, D. A. Wharton*, and P. J. Bishop

    Department of Zoology, University of Otago, Dunedin, New Zealand
    *Corresponding author –
    david.wharton@stonebow.otago.ac.nz

    Abstract

    The overwintering strategy of Litoria ewingii in Otago, New Zealand, was studied under laboratory and field conditions. Microhabitat temperature measurements showed that the frogs were often exposed to subzero temperatures. In the laboratory, L. ewingii tolerated freezing for up to 6 hrs at 1°C, and after the completion of the freezing event (ca. 1 hr) at 2°C. Frogs frozen with insulation survived freezing for 12 hrs at 1°C. Frogs supercooled to 1.2 ± 0.1°C and 1.7 ± 0.3°C on wet and dry substrates respectively. L. ewingii tolerated up to 47.5% of its body water frozen. Plasma glucose levels and osmolality were not increased during freezing. It is concluded that L. ewingii cannot avoid freezing and is sufficiently freeze tolerant to survive the subzero temperatures encountered during winter in Otago.

    Keywords: Litoria ewingii, freeze tolerance, overwintering, glucose, ice content, supercooling.

     

     

    CryoLetters 28 (5), 359-376 (2007)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

    THE USE OF PHYSICAL AND VIRTUAL INFRASTRUCTURES FOR THE VALIDATION OF ALGAL CRYOPRESERVATION METHODS IN INTERNATIONAL CULTURE COLLECTIONS

    John G. Day1*, Maike Lorenz2, Thomas A. Wilding1, Thomas Friedl2, Keith Harding3,4, Thomas Pröschold1, Debra Brennan1, Julia Müller2, Lília M.A. Santos5, M. Fátima Santos5, Hugo C. Osório5, Raquel Amaral5, Alena Lukešová6, Pavel Hrouzek6, Martin Lukeš6, Josef Elster7, Jaromír Lukavský7, Ian Probert8,9, Mathew J. Ryan10 and Erica E. Benson3,4

    1Scottish Association for Marine Science, Dunbeg, Argyll, PA37 1QA, UK;
    2Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany;
    3Plant Conservation Group, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, UK;
    4Current address: Damar, Research Scientists, Conservation, Environmental Science & Biotechnology, Drum Road, Cuparmuir, Cupar, Fife, KY15 5RJ, UK;
    5Instituto do Mar, Departamento de Botânica, Algoteca de Coimbra, Universidade de Coimbra, 3000, Coimbra, Portugal;
    6Institute of Soil Biology, Biology Centre ASCR, v.v.i., Na Sádkach 7, 370 05, České Budějovice, Czech Republic;
    7Institute of Botany AS CR, Dukelska 135, CZ 379 82 Třeboň, Czech Republic; 8Algobank, Université de Caen, CAEN cedex 14032, France;
    9Current address: Station Biologique, Place Georges Teissier, BP74, 29682 ROSCOFF Cedex, France;
    10CABI Europe - UK, Bakeham Lane, Egham, Surrey, TW20 9TY, UK.

    Abstract

    Two cryopreservation methods, colligative cryoprotection coupled with controlled cooling and vitrification-based, encapsulation-dehydration were validated by five members of the EU Research Infrastructure consortium, COBRA, and two independent external validators. The test strain Chlorella vulgaris SAG 211-11b was successfully cryopreserved using two-step cooling employing passive (Mr Frosty®) and Controlled Rate Freezers (CRF) attaining the desired recovery target within 15% of the median viability level (94%). Significant differences (P<0.05) between cooling regimes were observed where Mr Frosty® was more variable (Inter-Quartile Range being 21.5%, versus 13.0% for CRF samples). Viability assessment using fluorescein diacetate gave significantly (P<0.0001) higher survival than growth in agar with median values being 96% and 89%, respectively. On employing encapsulation-dehydration, greater variability between some validators was observed, with six labs observing recovery in 100% of the beads (84-95% of cells surviving) and one lab observing survival in 80% of the treated beads. Bead disruption followed by algal growth in agar was considered the most reliable and accurate method of assessing cell survival for encapsulation-dehydration.

    Keywords: cryopreservation, storage, culture collections, microalgae, protocol validation

     

     

    CryoLetters 28 (5), 377-386 (2007)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

    FIELD DEVELOPMENT OF OIL PALMS (ELĆIS GUINEENSIS JACQ.) ORIGINATING FROM CRYOPRESERVED STABILIZED POLYEMBRYONIC CULTURES (SPCS)

    E.K. Konan1, T. Durand-Gasselin2, Y.J. Kouadio3, A.C. Niamké1, D. Dumet4, 5, Y. Duval6, A. Rival7 and F. Engelmann 4, 8 *

    1Centre National de Recherche Agronomique (CNRA), Programme palmier, Laboratoire de culture in vitro – Station de recherche de La Mé, 13 BP 989 Abidjan 13, Côte d'Ivoire.
    2Centre de Coopération Internationale en Recherche Agronomique pour le Développement
    (CIRAD), Département BioS, UPR 28, TA 80/01, 34398 Montpellier cedex 5, France.
    3Laboratoire de Biologie et Amélioration des Productions Végétales, UFR des Sciences de la Nature, Université d'Abobo – Adjamé, 02 BP 801 Abidjan 02, Côte d'Ivoire.
    4Institut de Recherche pour le Développement (IRD), UMR DIA-PC, BP 64501, 34394 Montpellier cedex 5, France.
    (* correspondence:
    florent.engelmann@mpl.ird.fr).
    5International Institute of Tropical Agriculture (IITA), Oyo Road, PMB 5320, Ibadan, Nigeria (present address).
    6IRD, UMR DAP, BP 64501, 34394 Montpellier cedex 5, France.
    7CIRAD, Département BioS, UMR DAP, BP 64501, 34394 Montpellier cedex 5, France.
    8Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.

    Abstract

    In this paper, the long term observation of plants originating from control and cryopreserved stabilized polyembryonic cultures (SPCs) of six elite oil palm clones was carried out. Survival of plantlets in the nursery was monitored, then a series of vegetative and floral characteristics of over 440 palms were studied for up to 12 years after field transfer in Côte d'Ivoire. The six clones tested showed an average recovery of 34% after freezing in liquid nitrogen. The average survival in the nursery of plantlets originating from pretreated and dehydrated and from cryopreserved SPCs was higher than that of control SPCs. Palm trees originating from control SPCs were found to flower earlier than those originating from pretreated and dehydrated and from cryopreserved SPCs. This delay in flowering disappeared progressively and all palms had flowered 3 years after planting regardless of the SPC treatment. Abnormal palms were observed in one clone only. With this clone, the percentage of abnormal palms originating from cryopreserved SPCs was significantly lower (5%) than that measured on palms originating from control SPCs (29%).

    Keywords: cryopreservation; oil palm; stabilized polyembryonic cultures; flowering; field plant development.

     

     

    CryoLetters 28 (5), 387-399 (2007)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

    CRYOPRESERVATION OF EMBRYONIC AXES OF SELECTED AMARYLLID SPECIES

    Sershen, N.W. Pammenter*, Patricia Berjak and James Wesley-Smith

    Department of Biological and Conservation Sciences, University of KwaZulu-Natal, Durban, South Africa, 4041.
    *E-mail
    pammente@ukzn.ac.za

    Abstract

    A study on cryopreservation of excised embryonic axes of fifteen species of the Amaryllidaceae is reported. Embryonic axes that after flash-drying had a water content in the range 0.4 to 0.1 g g-1 and survival 60% were selected for cryopreservation procedures. The highest post-thaw viabilities (roots and shoots produced) across all species were recorded for embryonic axes subjected to rapid rather than slow cooling. With rapid cooling and no cryoprotection, the highest post-thaw viabilities for the fifteen species investigated was 0% in one species; ranged between 10 and 35% for nine species; and between 45 and 55% for five species. With cryoprotection and rapid cooling the highest post-thaw viabilities for these fifteen species was 0% for one species; ranged between 15 and 35% for six species; and between 40 and 75% for eight species. The highest post-thaw survival in ten out of fifteen species was obtained for axes dried to between 0.240.06 and 0.140.08 g g-1 (and rapidly cooled). With only one exception (Strumaria discifera; 45%), post-thaw survival after slow cooling ranged between 10 and 30%. Survival after vitrification plus slow cooling was achieved for seven species but was never higher than post-thaw survival in non-cryoprotected, rapidly cooled axes. The results suggest that species within the same family can exhibit commonalities in terms of amenability to cryopreservation techniques but for maximum success, axis water content and cooling rate particularly, must be optimised for each species in the family independently.

    Keywords: Amaryllidaceae, embryonic axes, cryoprotection, cooling rate

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