CryoLetters

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

CryoLetters 25, 375-388 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPROTECTANTS: THE ESSENTIAL ANTIFREEZES TO PROTECT LIFE IN THE FROZEN STATE

Barry J Fuller

University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, UK

Abstract

In the fifty years since the establishment of the cryoprotective effect of glycerol, cell banking by cryopreservation has become routine in many areas of biotechnology and medicine. Cryoprotectant addition has become a rather mundane step within the overall protocol. However, for future advances in cryobiology and to meet new challenges in the clinical use of cryopreserved cells or tissues, it will be essential to have an understanding of the development and current status of the biological and chemical knowledge on cryoprotectants (CPA). This review was undertaken to outline the history of CPA use, the important properties of CPA in relation to freezing damage, and what can be learnt from natural freezing-tolerant organisms. The conflicting effects of protection and toxicity resulting from use of CPA are discussed, and the role of CPA in enhancing ‘glassy’ states in the emerging field of vitrification are also set out.

Keywords: cryoprotectant; cryoprotectant toxicity; natural cryoprotectants; ice nucleating agents; antifreeze proteins; vitrification

CryoLetters 25, 389-404 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

FREEZE-DRYING OF PROTEIN PHARMACEUTICALS – THE APPLICATION OF THERMAL ANALYSIS

Vicky Kett¹*, Debra McMahon^1,2 & Kevin Ward²

¹School of Pharmacy, Queen’s University of Belfast, 97 Lisburn Road, Belfast, BT9 7BL
²Biopharma Technology Ltd., Winnall Valley Road, Winchester, SO23 0LD

Abstract

This review describes the range of thermal analysis techniques that may be used to investigate the key thermal properties of formulations to be freeze-dried & the resultant products. The use of these allows development of cost-effective processing while maintaining optimum product quality.

Keywords: thermal analysis, freeze-drying, glass transition, cryoprotectant, protein, lyophilization

CryoLetters 25, 405-414 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

AN IMPROVED CRYOPRESERVATION PROTOCOL FOR PINEAPPLE APICES USING ENCAPSULATION-VITRIFICATION

R. Gámez-Pastrana¹, Y. Martínez-Ocampo¹, C.I. Beristain² and M.T. González-Arnao ^2,3*

¹ Universidad Veracruzana, Facultad de Ciencias Biológicas y Agropecuarias, Córdoba, Veracruz, México.
² Universidad Veracruzana, Instituto de Ciencias Básicas, Apartado Postal 575, C.P. 91000, Xalapa, Veracruz, México.
³ On leave from Universidad de La Habana, Facultad de Biología, Ciudad de La Habana, Cuba. (E-mail: mtgarnao@ivia.es).
* To whom correspondence should be addressed.

Abstract

Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0°C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 %) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in    0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0°C for 60 min before immersion into liquid nitrogen.  Following this procedure     54 % and 83 % of apices from MD-2 and Puerto Rico varieties respectively survived.

Keywords: vitrification, encapsulation-vitrification, Plant Vitrification Solutions (PVS), pineapple, dehydration, cryopreservation.

CryoLetters 25, 415-424 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

STUDIES ON CRYOPROTECTANT TOXICITY TO ZEBRAFISH (Danio rerio) OOCYTES

M. Plachinta, T. Zhang* and D.M. Rawson

Luton Institute of Research in the Applied Natural Sciences, The Spires, 2 Adelaide Street, Luton, Bedfordshire, LU1 5DU, United Kingdom
*For correspondence: tiantian.zhang@luton.ac.uk

Abstract

Cryopreservation of fish germ cells is an important measure in conservation of fish genetic material. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. In the present study the toxicity of cryoprotectants to zebrafish (Danio rerio) oocytes was investigated. Commonly used cryoprotectants dimethyl sulfoxide (DMSO), methanol, ethylene glycol (EG), propylene glycol (PG), sucrose and glucose were studied. Stage III (vitellogenic), stage IV (maturation) and stage V (mature egg) zebrafish oocytes were incubated in Hank's medium containing different concentrations of cryoprotectants (0.25-4M) for 30min at room temperature. Three different tests were used to assess oocyte viability: trypan blue (TB) staining, thiazolyl blue (MTT) staining and in vitro maturation followed by observation of germinal vesicle breakdown (GVBD). Results showed that the toxic effect of cryoprotectant on oocytes generally increased with increasing concentration. MTT test was shown to be the least sensitive testing method and gave poor correlation to subsequent GVBD results. Sensitivity of vital tests increases in the order of MTT, TB and GVBD. GVBD test showed that cryoprotectant toxicity to stage III zebrafish oocytes increased in the order of methanol, PG, DMSO, EG, glucose and sucrose. No Observed Effect Concentrations (NOECs) for stage III oocytes were 2M, 1M, 1M, 0.5M, <0.25M and <0.25M for methanol, PG, DMSO, EG, glucose and sucrose respectively. TB test also showed that the toxicity of tested cryoprotectants increased in the same order. The sensitivity of oocytes to cryoprotectants appeared to increase with development stage with stage V oocytes being the most sensitive.

Keywords: zebrafish (Danio rerio), oocytes, toxicity, cryopreservation, cryoprotectants

CryoLetters 25, 425-434 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

COLLAPSE TEMPERATURE OF BACTERIAL SUSPENSIONS: THE EFFECT OF CELL TYPE AND CONCENTRATION

Fernanda Fonseca^*, Stéphanie Passot, Pascale Lieben and Michèle Marin

UMR Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France.
^* Corresponding author : Tel.: (33) (0) 1 30 81 59 40. Fax: (33) (0) 1 30 81 55 97.
E-mail address: fonseca@grignon.inra.fr

Abstract

The characterisation of the physical state of frozen and freeze dried biological products delivers powerful information for freeze-drying process optimisation. The influence of lactic acid bacterial cell size, shape and concentration on collapse temperature of concentrated bacterial suspensions was investigated. Lactobacillus bulgaricus (long rods), and Streptococcus thermophilus (small spherical cells) were used as cellular models for this study. Whatever the strain, when lactic acid bacterial cells were added to protective solutions, the collapse temperature increased, thus allowing the use of higher sublimation temperatures during primary drying than expected from the protective medium alone. Moreover, the higher the cell concentration, the greater the effect, linear relationships existing between the collapse temperatures and the total dried matter. Cells of both strains gave a kind of robustness to the freeze-dried product, but the increase observed in collapse temperature was considerably higher (3-5°C) for L. bulgaricus compared to S. thermophilus. This result was ascribed to the different size and shape of the strains.

Keywords: collapse temperature, glass transition temperature, lactic acid bacteria, freeze-drying optimisation

Abstracts

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