John G. Day

Cryopreservation and freeze-drying Protocols

Contents Preface Contributors Chapter 1 Long-term ex situ conservation of Biological Resources and the role of Biological Resource Centres Glyn N. Stacey and John G. Day Chapter 2 The Process of Freeze-drying Gerald Adams Chapter 3 Principles of Cryopreservation David E. Pegg Chapter 4 Lyophilization of Proteins Paul Matejschuk Chapter 5 Vacuum-drying and Cryopreservation of Prokaryotes Brian J. Tindall Chapter 6 Freeze-drying of Yeast Cultures Chris Bond Chapter 7 Cryopreservation of Yeast Cultures Chris Bond Chapter 8 Freeze-drying Fungi using a Shelf-freeze-drier C. Shu-hui Tan, Cor W. van Ingen and Joost A. Stalpers Chapter 9 Cryopreservation and Freeze-drying of Fungi employing Centrifugal and Shelf Freeze-drying Matthew J. Ryan and David Smith Chapter 10 Cryopreservation of Microalgae and Cyanobacteria John G. Day Chapter 11 Cryopreservation of Plant Cell Suspensions Brian W. W. Grout Chapter 12 Cryopreservation of Shoot-tips and Meristems Erica E. Benson, Keith Harding and Jason W. Johnston Chapter 13 Cryopreservation of Desiccation Tolerant Seeds Hugh W. Pritchard Chapter 14 Cryopreservation of Fish Sperm Eugeny Kopeika, Julia Kopeika and Tiantian Zhang Chapter 15 Cryopreservation of Avian Spermatozoa Graham Wishart Chapter 16 Cryopreservation of Animal and Human Cell Lines Christopher B. Morris Chapter 17 Cryopreservation of Haematopoietic Stem/Progenitor Cells for Therapeutic Use Suzanne M. Watt, Eric Austin and Sue Armitage Chapter 18 Cryopreservation of Human Embryonic Stem Cell Lines Charles J. Hunt and Paula M. Timmons Chapter 19 Cryopreservation of Primary Animal Cell Cultures Glyn N. Stacey and Stuart Dowall Chapter 20 Cryopreservation of Red Blood Cells and Platelets Andreas Sputtek Chapter 21 Cryopreservation of Mammalian Semen Mark R. Curry Chapter 22 Cryopreservation of Mammalian Oocytes Sharon J. Paynter and Barry J. Fuller Chapter 23 Cryopreservation of Mammalian Embryos Barry J. Fuller and Sharon J. Paynter Glossary

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 16S rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204T), Planktothrix rubescens (type strain CCAP 1459/22T), Planktothrix pseudagardhii sp. nov. (type strain T1-8-4T), Planktothrix mougeotii (type strain TR1-5T), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207T), Tychonema bourrellyi (type strain CCAP 1459/11BT) and Limnothrix redekei (type strain NIVA CYA 277/1T).

Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy

Commercial success of algal-based biofuels depends on growth characteristics and lipid metabolism of the production species. The oleaginous microalgae, Thalassiosira pseudonana, Odontella aurita, Nannochloropsis oculata, Isochrysis galbana, Chromulina ochromonoides, and Dunaliella tertiolecta, were cultivated under a matrix of two temperatures (10 and 20 °C) and two nutrient regimes (deplete and replete). For all species, a strong negative correlation between growth rate and lipid content was observed. Multiple stressors have no additive effect on lipid accumulation. Total oil content (fatty acid methyl esters, FAMEs, pg cell(-1)) was increased more by nutrient limitation than by temperature stress. In response to nutrient stress, N. oculata emerged as the most robust species with an increase in lipid accumulation of up to three to four-fold compared to the accumulation under nutrient sufficient conditions. Although stress conditions led to reduced fatty acid unsaturation in most taxa due to increased triacylglycerol (TAG) production, a high proportion of eicosapentaenoic acid (EPA) was maintained in O. aurita.

Overcoming biological constraints to enable the exploitation of microalgae for biofuels

Microalgae have significant potential to form the basis of the next biofuel revolution. They have high growth and solar energy conversion rates. Furthermore, their osmotolerance, metabolic diversity and capacity to produce large amounts of lipids have attracted considerable interest. Although there are a handful of commercially successful examples of the photoautotrophic mass-culture of algae, these have focused on the production of higher value products (pigments, health-foods etc.). The technical and commercial challenges to develop an economically viable process for biofuels are considerable and it will require much further R&D. In this paper the biological constraints, with a particular focus on strain selection are discussed.

Cryopreservation-recalcitrance in microalgae: novel approaches to identify and avoid cryo-injury.

Standard two-step freezing protocols areunsatisfactory for Euglena gracilis and manyother microalgae, particularly those with larger cellsizes, complex morphologies and/or those susceptibleto environmental stress. Using techniques that allowmechanisms of injury and sites of damage to beidentified (e.g. monitoring oxygen evolving capacity,detection of •OH, microscopic visualisation ofintracellular ice and structural/ultrastructuraldamage), it is possible to improve conventionalcryopreservation methodologies. In E. gracilisthis has resulted in the development of protocolswhich increased post-thaw viability levels from 0 to20%. Alternative cryoprotection strategies testedincluded vitrification and encapsulation/dehydration.Vitrification was unsuccessful due to the hightoxicity of the solutions. Encapsulation/dehydration,with or without two-step cooling were suitable forcryopreservation of E. gracilis, the latterresulted in the highest levels of post-thaw viability(40%) and viability was maintained after 12 monthsstorage.

Studies of Free Radical-Mediated Cryoinjury in the Unicellular Green Alga Euglena gracilis Using a Non-Destructive Hydroxyl Radical Assay: A Novel Approach for Developing Protistan Cryopreservation Strategies

The development of cryoconservation methods for the long-term storage of algal cultures is important for the ex situ preservation of biological diversity and the maintenance of genetic stability within this group of important organisms. However, as many unicellular algae are recalcitrant to cryogenic storage, this study aims to evaluate the role of oxidative stress in cryoinjury. A non-invasive, non-destructive assay method previously applied to animal cells has been developed to evaluate free radical mediated oxidative stress in Euglena gracilis exposed to different cryopreservation treatments. The procedure employs dimethyl sulphoxide as a probe for the hydroxyl radical. Adopting this approach it was possible to identify those components of the cryopreservation protocol which were the most damaging. These were identified as preparative centrifugation and sub-zero freezing treatments. Post-storage survival in E. gracilis was significantly (P < 0.05) enhanced when the chelating agent desferrioxamine was included in the recovery medium whilst methane production was significantly (P < 0.004) reduced, suggesting that the additive was capable of ameliorating oxidative stress. The potential of using novel, exogenous antioxidant treatments developed for medical applications and applying them to enhance cryopreservation tolerance in recalcitrant unicellular algae is discussed.

In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

SummaryMicroalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.

Distinction between multiple isolates of Chlorella vulgaris (Chlorophyta, Trebouxiophyceae) and testing for conspecificity using amplified fragment length polymorphism and its rDNA sequences

Multiple strains of individual algal species are available from public culture collections, often with the same isolate being maintained in parallel at a number of collections under different culture regimes. To unravel genomic variation and to identify unique genotypes among such multiple strains, two approaches were used on a sample of 29 strains of Chlorella vulgaris Beijerinck, an alga of great value for applied research, from five culture collections. With the exception of two strains, internal transcribed spacer rDNA sequence data substantiated conspecificity of the studied strains and only minor sequence differences with the authentic “Beijerinck isolate” were observed. Amplified fragment length polymorphism (AFLP) detected considerable genomic variation when rDNA sequences were identical. Band detection and the construction of a binary matrix from AFLP patterns for phylogenetic analyses were fully automated, but comparison of similar patterns still required manual refinement. The AFLPs distinguished 11 unique genotypes and provided robust support for the presence of five cryptic species. This finding advocates the need to carefully record which strain has been used in any experiment or in applied research, because genomic variation may also correspond to differences in physiological/biochemical properties. No genomic differences could be detected between duplicate strains of the same isolate that were maintained by continuous subculturing over many decades or within those stored at ultralow temperatures.

An investigation of the heterotrophic culture of the green algaTetraselmis

The marine PrasinophyteTetraselmis may be cultured under both mixotrophic (photoheterotrophic) and heterotrophic conditions. The growth rate was slightly lower, and pigment levels and lipid composition were radically affected on heterotrophic culture in 1 L fermenters. Total chlorophyll levels of dark grown cultures were less than 1% of those observed in mixotrophically grown cells, the chlorophylla : b ratio also decreased as did the carotenoid content. In addition, the total amounts of lipids including polyunsaturated fatty-acids were also lower in heterotrophically cultured cells: 6.4 mg g−1 (dried alga) and 0.35 mg g−1 (dried alga); as compared to 37.1 mg g−1 (dried alga) and 18.5 mg g−1 (dried alga), for cells grown in the light. However, gross morphology and final yield (>16 g l−1) were relatively unaffected. The algae produced were spray-dried and tested for their suitability as an aquaculture feed.

Distinction of isolates among multiple strains of Chlorella vulgaris (Chlorophyta, Trebouxiophyceae) and Testing Conspecificity with Amplified Fragment Length Polymorphism and ITS RDNA sequences. J. Phycol. 41, 1.

Multiple strains of individual algal species are available from public culture collections, often with the same isolate being maintained in parallel at a number of collections under different culture regimes. To unravel genomic variation and to identify unique genotypes among such multiple strains, two approaches were used on a sample of 29 strains of Chlorella vulgaris Beijerinck, an alga of great value for applied research, from five culture collections. With the exception of two strains, internal transcribed spacer rDNA sequence data substantiated conspecificity of the studied strains and only minor sequence differences with the authentic “Beijerinck isolate” were observed. Amplified fragment length polymorphism (AFLP) detected considerable genomic variation when rDNA sequences were identical. Band detection and the construction of a binary matrix from AFLP patterns for phylogenetic analyses were fully automated, but comparison of similar patterns still required manual refinement. The AFLPs distinguished 11 unique genotypes and provided robust support for the presence of five cryptic species. This finding advocates the need to carefully record which strain has been used in any experiment or in applied research, because genomic variation may also correspond to differences in physiological/biochemical properties. No genomic differences could be detected between duplicate strains of the same isolate that were maintained by continuous subculturing over many decades or within those stored at ultralow temperatures.