Browsing by Author "Harding, Keith"

Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.

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.

An encapsulation/dehydration procedure was developed for Euglena gracilis Klebs as a 'model alga' to examine various cryoprotective regimes combined with controlled rate cooling to cryopreserve other Euglenoid taxa. Cryoprotective variables were optimised to enable reproducible growth following a combination of alginate encapsulation, sucrose osmotic dehydration, air desiccation, methanol treatment, cooling to -40 degrees C and plunging into liquid nitrogen (LN). Amplified Fragment Length Polymorphism (AFLP) analysis was adapted to: (i) verify algal identity by discriminating between different Euglenoids and (ii) examine the genetic stability of algal cultures prior to various stages of cryoprotective treatments and following exposure to LN. AFLPs were highly reproducible (>99%) as reliable diagnostic markers, where a single DNA fragment change accounted for similar to 0.4% of the detectable variation in an AFLP pattern. AFLP changes were detected in cryoprotective treatments following LN exposure. Successive stages of the dehydration and desiccation treatments did not accumulate AFLP changes indicating these are random events.

This review pertains to a project (COBRA QLRT-2000-01645, http://www.cobra.ac.uk) supported by the European Commission's Fifth Framework Programme for Quality of Life and Management of Living Resources. The aim of this initiative is to develop and apply novel cryo-preservation protocols to microalgae and cyanobacteria that are difficult to cryopreserve. One such approach utilizes cryoprotective vitrification for the conservation of algal culture collections at -196degreesC in liquid nitrogen. Achieving a vitrified state depends on increasing cellular viscosity to a critical point at which water forms an amorphous, non-crystalline glass on cooling to ultra-low temperatures. Vitrification has been utilized extensively in medical cryobiology and for the preservation of animal, human and higher plant germplasm but its application for the conservation of algae and cyarlobacteria is less widespread. The review provides a theoretical introduction to vitrification and highlights current and potential applications in algal preservation, especially for storage-recalcitrant organisms.

The first version of the Standard PREanalytical Code (SPREC) was developed in 2009 by the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group to facilitate documentation and communication of the most important preanalytical quality parameters of different types of biospecimens used for research. This same Working Group has now updated the SPREC to version 2.0, presented here, so that it contains more options to allow for recent technological developments. Existing elements have been fine tuned. An interface to the Biospecimen Reporting for Improved Study Quality (BRISQ) has been defined, and informatics solutions for SPREC implementation have been developed. A glossary with SPREC-related definitions has also been added.

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((R))) 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((R)) 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.