Alginate Encapsulation to Enhance Biopreservation Scope and Success: A Multidisciplinary Review of Current Ideas and Applications in Cryopreservation and Non-Freezing Storage.

BACKGROUND: The development of encapsulation technologies has played an important role in improving cryopreservation outcomes for many cell and tissue types over the past 20 years. Alginate encapsulation cryopreservation (AECryo) has been incorporated into a range of applications in biotechnology, species conservation and clinical therapies, using cells from many different phyla, including higher plants, animal and human cells. This review describes the background to the origins of AECryo, the development of AECryo in higher plant tissues, broadening to current applications in algal conservation, the roles for AECryo in preserving phytodiversity, fungal species and in animal and human cells. OBJECTIVE: The main aims are to provide information resources on AECryo in different areas of biology and to stimulate new ideas for wider applications and future improvement. The translation of this useful biopreservation strategy into new opportunities for cell cryopreservation and storage at non-freezing temperatures are also discussed.

Comparative Biology of Cycad Pollen, Seed and Tissue - A Plant Conservation Perspective.

Cycads are the most endangered of plant groups based on IUCN Red List assessments; all are in Appendix I or II of CITES, about 40% are within biodiversity 'hotspots,' and the call for action to improve their protection is long-standing. We contend that progress in this direction will not be made until there is better understanding of cycad pollen, seed and tissue biology, which at the moment is limited to relatively few (<10%) species. We review what is known about germplasm (seed and pollen) storage and germination, together with recent developments in the application of contemporary technologies to tissues, such as isotype labelling, biomolecular markers and tissue culture. Whilst progress is being made, we conclude that an acceleration of comparative studies is needed to facilitate the integration of in situ and ex situ conservation programmes to better safeguard endangered cycads.

A life cycle model to enable research of cryostorage recalcitrance in temperate woody species: the case of sitka spruce (Picea sitchensis).

Empirical testing of protocols and fundamental investigations are the approaches usually applied to study germplasm storage recalcitrance in temperate plants. However, they can fall short of practicable solutions, even after exhaustive experimentation, and the generation of negative survival data makes it difficult to plan further investigations. Picea sitchensis somatic embryos are amenable to cryopreservation whereas in vitro shoot meristems, although able to survive, are incapable of sustained recovery. Differential Scanning Calorimetry (DSC) revealed that these disparate responses could not be attributed to biophysical factors. A model is presented hypothesising that in some cases life cycle adaptations (cold hardening, dormancy) may have opposing influences on survival causing delayed-onset, cryogenically-induced loss of viability in temperate tree species.

Developing seed cryobank strategies for Tabebuia heptaphylla (Bignoniaceae), a hardwood tree of the Brazilian South Atlantic Forest.

The conservation of Tabebuia heptaphylla, an economically significant, endangered tree of the South Atlantic Forest is confined to arboreta. Although its seeds are orthodox, they do not withstand long-term storage in conventional seed banks, motivating the development of cryopreservation for this species. Seeds within the moisture content (MC) range of 7.5 percent (0.08 g water g dry mass) to 8.4 percent (0.09 g water g dry mass) germinated after storage in liquid nitrogen (LN). Storage duration (15 min to 26 weeks) and rewarming regime (slow and rapid) did not significantly influence germination, which ranged between 54-67 percent. As no additional cryoprotective treatments were required, the protocol is time-, cost- and technically-efficient. Because transport of seeds in LN is problematic for safety, logistic and technical reasons, the feasibility of implementing germplasm transfer using T. heptaphylla seeds recovered from cryobanks was also tested. Viability was not negatively affected in seeds that had been rewarmed, recovered and maintained at room temperature for 2 weeks, allowing safe germplasm transfer in the unfrozen state. The vigor of seedlings from cryopreserved seeds, which was evaluated 90 days after transfer to soil was not influenced by LN storage compared to the controls.

Detection of 8-hydroxy-2-deoxyguanosine as a marker of oxidative damage in DNA and germplasm exposed to cryogenic treatments.

An HPLC method has been optimised to measure 8-hydroxy-2-deoxyguanosine (8OHdG) in DNA and germplasm with the objective of using the adduct as a marker of cryostorage stability. The encapsulation-dehydration cryopreservation protocol was adapted as a model system for assessing the formation of 8OHdG from alginate-encapsulated DNA of calf thymus (CT) and currant species (Ribes) exposed to temperatures of -20 and -196 degree C. The presence of H2O2 exacerbated the formation of 8OHdG in encapsulated CT and Ribes DNA. Production of the oxidized adduct was lower in the plant system. A reduction in residual water following osmotic dehydration and evaporative desiccation was associated with reduced adduct formation in encapsulated DNA. No significant differences in 8OHdG adduct formation were observed in plants regenerated from cryopreserved Ribes meristems derived from genotypes known to have differential tolerance to cryopreservation.

Deployment of the encapsulation-dehydration protocol to cryopreserve microalgae held at the Sammlung von Algenkulturen, Universitat Gottingen, Germany.

Encapsulation-dehydration was applied to cryopreserve 14 diverse algal strains, representing eukaryotic terrestrial microalgae; of these 12 survived to form cell colonies after recovery from cryostorage. Surviving algae had varying degrees of tolerance to osmotic dehydration and desiccation in this vitrification-based cryoprotective strategy. The extent of algal regrowth was affected by the mode of desiccation (silica gel or air-flow), the duration of evaporative desiccation and exposure to light during early recovery phase. This paper: (i) demonstrates the versatility of the encapsulation/dehydration method to cryopreserve diverse microalgae; (ii) confirms the successful transfer of this cryostorage technology to the Culture Collection of Algae at Gottingen University (SAG); and (iii) recommends encapsulation/dehydration as a feasible alternative to controlled rate cooling for preserving algae held in international culture collections.

Cryopreservation and conservation of microalgae: the development of a Pan-European scientific and biotechnological resource (the COBRA project).

Microalgae are one of the most biologically important elements of worldwide ecology and could be the source of diverse new products and medicines. COBRA (The COnservation of a vital european scientific and Biotechnological Resource: microAlgae and cyanobacteria) is the acronym for a European Union, RTD Infrastructures project (Contract No. QLRI-CT-2001-01645). This project is in the process of developing a European Biological Resource Centre based on existing algal culture collections. The COBRA project's central aim is to apply cryopreservation methodologies to microalgae and cyanobacteria, organisms that, to date, have proved difficult to conserve using cryogenic methods. In addition, molecular and biochemical stability tests have been developed to ensure that the equivalent strains of microorganisms supplied by the culture collections give high quality and consistent performance. Fundamental and applied knowledge of stress physiology form an essential component of the project and this is being employed to assist the optimisation of methods for preserving a wide range of algal diversity. COBRA's "Resource Centre" utilises Information Technologies (IT) and Knowledge Management practices to assist project coordination, management and information dissemination and facilitate the generation of new knowledge pertaining to algal conservation. This review of the COBRA project will give a summary of current methodologies for cryopreservation of microalgae and procedures adopted within the COBRA project to enhance preservation techniques for this diverse group of organisms.

Oxidative stress in recalcitrant tissue cultures of grapevine.

Thiobarbituric acid reactive substances (TBARS), and fluorescent compounds with spectral characteristics typical of products associated with oxidative stress in senescent and aging plant and animal cells, were detected in tissue cultures of the recalcitrant grapevine Vitis vinifera L. cultivar, Sultanina. These compounds increased during the early stages of dedifferentiation (callogenesis) of nodal stem explants. Catalase activity was not detected in the original explant, but was induced during callogenic dedifferentiation. Conversely, superoxide dismutase activity was detectable in the original explant, but diminished during the first week of callus induction. Transfer to callus induction medium promoted a large increase in the sulfhydryl content of nodal tissues. TBARS and fluorescent products accumulated in Sultanina callus during long-term culture (over 6 months). The possibility that oxidative stress may contribute to culture recalcitrance in this vine is discussed.

Malondialdehyde and 4-hydroxy-2-nonenal in plant tissue cultures: LC-MS determination of 2,4-dinitrophenylhydrazone derivatives.

The cytologically active secondary lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) have been detected as their 2,4-dinitrophenylhydrazone (DNP) derivatives in plant tissue cultures using LC-MS. This paper reports, for the first time, the use of LC-MS methodology to definitively identify 4-hydroxy-2-nonenal in plants. Limits of detection for the two derivatives are approximately 5 pmol (1.2 x 10(-9) g; 1 microM) and 0.1 pmol (3 x 10(-11) g; 20 nM) respectively. Mass spectrometer response was linear in the range from 2-200 microM DNP-MDA and 0.02-10 microM DNP-HNE. This methodology has been used to assess the formation of aldehydic secondary lipid peroxidation products in dedifferentiated callus cultures of Daucus carota. The finding that profiles of MDA and HNE can be correlated with embryogenic competence is of considerable interest as oxidative status has already been implicated as a regulatory factor in animal development.

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. Poststorage 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.

The use of microsatellite analysis in Solanum tuberosum l. in vitro plantlets derived from cryopreserved germplasm.

This study reports the application of the encapsulation/dehydration cryopreservation and microsatellite analysis to the conservation of Solanum tuberosum cultivars Brodick and Golden Wonder. Cryopreserved shoot-tips were capable of up to 40% shoot and plantlet regeneration in Brodick and >60 % for Golden Wonder. Microsatellite analysis was used with genomic DNA of Golden Wonder and Desiree to establish DNA sequence length polymorphisms. As the basis of stability assessments this technique was applied to: (i) individual, field-grown, plants of Golden Wonder; (ii) individual Golden Wonder plants derived from a single parental tuber progeny; (iii) plantlets derived from in vitro cultures of Golden Wonder and Brodick and (iv) Golden Wonder and Brodick plantlets derived from cryopreserved germplasm

Profiling cryopreservation protocols for Ribes ciliatum using differential scanning calorimetry.

DSC analysis was performed at three points in the cryopreservation process on encapsulated-dehydrated meristems of Ribes ciliatum. Meristems were excised from shoots pre-treated with either sucrose or glucose, encapsulated in alginate beads, dehydrated in sucrose solutions, air dried, and plunged in liquid nitrogen. Thermal analysis revealed glass transitions during cooling of air-desiccated meristems, however, on rewarming a small endothermic event was detected suggesting glass destabilization can occur. Interestingly, this did not occur in alginate beads or meristems when these components were cooled and rewarmed separately. The possibility exists that thermal and moisture gradients may arise within the alginate bead/tissue complex and we propose that the heterogeneous composition of the meristems and the surrounding alginate may promote ice nucleation on rewarming. The significance of this regarding the stabilization of glasses formed in alginate beads and their encapsulated meristems is discussed. This study also reports an approach to Ribes cryopreservation in which the pregrowth of shoots in 0.75M sucrose for 1 week can be used as a substitute for cold acclimation.

Variation in free-radical damage in rice cell suspensions with different embryogenic potentials.

Levels of free-radical-mediated lipid peroxidation were monitored in cell-suspension cultures of Oryza sativa L. possessing different embryogenic potentials. Oxidative stress was evaluated using assays which sequentially assessed the stages of lipid peroxidation (diene conjugation, peroxidation, and the formation of secondary lipid-peroxidation products). Lipid peroxidation was significantly higher in a cell line which had lost embryogenic ability compared with lines which still retained this capacity. Superoxide dismutase (EC 1.15.1.1) activity did not vary significantly between the embryogenic and previously embryogenic lines; however, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) activities were significantly lower in the line which had lost embryogenic ability. Metabolic activity as estimated by reduction of triphenyl tetrazolium chloride decreased with diminishing embryogenic potential and was especially low in cell lines which never exhibited embryogenic capabilities. The possible involvement of free radicals in the loss of embryogenic potential of rice cells is discussed.

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow.

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.