"Life in Data"--Outcome of a Multi-Disciplinary, Interactive Biobanking Conference Session on Sample Data.

INTRODUCTION: Clinical, biodiversity, and environmental biobanks share many data standards, but there is a lack of harmonization on how data are defined and used among biobank fields. This article reports the outcome of an interactive, multidisciplinary session at a meeting of the European, Middle Eastern, and African Society for Biopreservation and Biobanking (ESBB) designed to encourage a 'learning-from-each-other' approach to achieve consensus on data needs and data management across biobank communities. MATERIALS, METHODS, AND RESULTS: The Enviro-Bio and ESBBperanto Working Groups of the ESBB co-organized an interactive session at the 2013 conference (Verona, Italy), presenting data associated with biobanking processes, using examples from across different fields. One-hundred-sixty (160) diverse biobank participants were provided electronic voting devices with real-time screen display of responses to questions posed during the session. The importance of data standards and robust data management was recognized across the conference cohort, along with the need to raise awareness about these issues within and across different biobank sectors. DISCUSSION AND CONCLUSION: While interactive sessions require a commitment of time and resources, and must be carefully coordinated for consistency and continuity, they stimulate the audience to be pro-active and direct the course of the session. This effective method was used to gauge opinions about significant topics across different biobanking communities. The votes revealed the need to: (a) educate biobanks in the use of data management tools and standards, and (b) encourage a more cohesive approach for how data and samples are tracked, exchanged, and standardized across biobanking communities. Recommendations for future interactive sessions are presented based on lessons learned.

Writing standard operating procedures (SOPs) for cryostorage protocols: using shoot meristem cryopreservation as an example.

Standard operating procedures are a systematic way of making sure that biopreservation processes, tasks, protocols, and operations are correctly and consistently performed. They are the basic documents of biorepository quality management systems and are used in quality assurance, control, and improvement. Methodologies for constructing workflows and writing standard operating procedures and work instructions are described using a plant cryopreservation protocol as an example. This chapter is pertinent to other biopreservation sectors because how methods are written, interpreted, and implemented can affect the quality of storage outcomes.

Cryopreservation of shoot tips and meristems: an overview of contemporary methodologies.

Cryopreservation is the storage of viable bioresources at ultra-low temperatures in liquid nitrogen (LN). This chapter provides an overview of those protocols most commonly used to cryopreserve in vitro derived shoot tips and meristems; they are described generically, as sequential technical steps, including preparative and cryogenic treatments and the morphogenetic assessment of recovery. The importance of translating research-generated methods into formal Standard Operating Procedures (SOPs) is considered.

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos.

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H(2)O(2) was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.

Standard PREanalytical Codes: A New Paradigm for Environmental Biobanking Sectors Explored in Algal Culture Collections.

The Standard PREanalytical Code (SPREC) was developed by the medical/clinical biobanking sector motivated by the need to harmonize biospecimen traceability in preanalytical processes and enable interconnectivity and interoperability between different biobanks, research consortia, and infrastructures. The clinical SPREC (01) consists of standard preanalytical variable options (7-code elements), which comprise published and (ideally) validated methodologies. Although the SPREC has been designed to facilitate clinical research, the concept could have utility in biorepositories and culture collections that service environmental and biodiversity communities. The SPREC paradigm can be applied to different storage regimes across all types of biorepository. The objective of this article is to investigate adapting the code in nonclinical biobanks using algal culture collections and their cryostorage as a case study. The SPREC (01) is recalibrated as a putative code that might be adopted for biobanks holding different types of biodiversity; it is extended to include optional coding from the point of sample collection to postcryostorage manipulations, with the caveat that the processes are undertaken by biorepository personnel.

Standard preanalytical coding for biospecimens: defining the sample PREanalytical code.

BACKGROUND: Management and traceability of biospecimen preanalytical variations are necessary to provide effective and efficient interconnectivity and interoperability between Biobanks. METHODS: Therefore, the International Society for Biological and Environmental Repositories Biospecimen Science Working Group developed a "Standard PREanalytical Code" (SPREC) that identifies the main preanalytical factors of clinical fluid and solid biospecimens and their simple derivatives. RESULTS: The SPREC is easy to implement and can be integrated into Biobank quality management systems and databases. It can also be extended to nonhuman biorepository areas. Its flexibility allows integration of new novel technological developments in future versions. SPREC version 01 is presented in this article. CONCLUSIONS AND IMPACT: Implementation of the SPREC is expected to facilitate and consolidate international multicenter biomarker identification research and biospecimen research in the clinical Biobank environment.

Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm.

The physiological and molecular mechanisms associated with acclimation and survival have been examined in four Ribes genotypes displaying differential cryotolerance. Changes in DNA methylation, nucleic acid and nucleoside composition were determined during acclimation and recovery of in vitro shoot-meristems from cryopreservation. DNA methylation was induced in the tolerant genotype, while demethylation was evident in sensitive genotypes. This response initially occurred during sucrose simulated acclimation, with progressive changes as shoots recovered from successive stages of the encapsulation-dehydration protocol. These methylation patterns existed in the initial vegetative cycle but regressed to control values following subculture, indicating the changes in DNA methylation to be a reversible epigenetic mechanism. RNA levels indicating transcriptional activity during the acclimation of nodal tissue are inversely linked to methylation changes, where activity appears to be up-regulated in the cryosensitive genotypes. Conversely, cryopreserved shoots show increased levels of both RNA and DNA methylation in the cryotolerant genotypes. Other nucleosides show post-transcriptional activity corresponds with tolerance during acclimation and cryopreservation. These observations connect physiological attributes to differential molecular changes in Ribes, the implications of which are discussed in relation to cryopreservation-induced apoptosis and genetic stability.

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds.

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.

Developing cryopreservation for Picea sitchensis (sitka spruce) somatic embryos: a comparison of vitrification protocols.

Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, approximately 10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P less than 0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation-dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.

Deployment of the encapsulation-dehydration protocol to cryopreserve polar microalgae held at the Czech Republic Academy of Sciences Institute of Botany.

Polar isolates of four chlorococcal microalgae originating from the Arctic and Antarctica withstand cryopreservation using encapsulation-dehydration. Viability assessments, which initially used chloroplhyll fluorescence (Kautsky) induction kinetics, revealed that all strains suffered photosynthetic impairment during early post-cryopreservation recovery. This cryoinjury was reversible, as indicated by cell regrowth in three of the four strains. Lack of growth in the fourth isolate was due to contaminating bacteria rather than cryogenic factors.

Deployment of the encapsulation-dehydration protocol to cryopreserve diverse microalgae held at the Institute of Soil Biology, Academy of Sciences of the Czech Republic.

Twenty-seven strains of soil algae isolated from highly diverse provenances and habitats were assessed for their capacity to withstand cryopreservation using encapsulation/dehydration. Survival was assessed following the release of algae from alginate beads treated with sodium hexametaphosphate and regrowth was assessed using NAJA Image Analysis. Regrowth occurred in 19 strains, with > 50% survival being observed in 15. Algal tolerance to osmotic dehydration and evaporative desiccation was critical to the success of the method. Recovery in five out of the remaining eight recalcitrant strains was enhanced by substituting sorbitol for the osmotic pretreatment or by combining encapsulation with two-step controlled rate cooling.

Cryopreservation of shoot tips and meristems.

Shoot-tip meristem cryopreservation methodologies are reported for the complementary cryoprotective strategies of vitrification and equilibrium freezing using traditional controlled-rate freezing and chemical additive cryoprotection. Pregrowth, pretreatment, and cold acclimation approaches for the improvement of tolerance to liquid nitrogen are also presented. The chapter concludes by reporting an analytical protocol that profiles volatile hydrocarbon stress markers (for ethylene, hydroxyl radicals, and lipid peroxidation products) during cryopreservation. This method uses noninvasive headspace sampling and gas chromatography, and it is widely applicable across cryogenic systems.

The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections.

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.

Cryopreservation of Picea sitchensis (sitka spruce) embryogenic suspensor masses.

A cryopreservation method comprising sorbitol pre-growth treatment, DMSO cryoprotection and two-step controlled rate cooling has been optimized for differentiated embryogenic suspensor masses (ESM) of Picea sitchensis. The protocol was applied to clonal cultures from five different half-sibling families each represented by five different genotypes and their responses to cryopreservation assessed over 3 years. Nineteen of the 25 clonal lines tested survived LN and were capable of regrowth and producing stage 2-4 somatic embryos. Following the second subculture cycle of esm after they had been retrieved from cryogenic storage, post-cryopreservation regrowth was comparable with that of controls. A general linear model, multifactorial main effects plot revealed no significant differences between genotypes (p > 0.05) in response to cryopreservation, whereas significant differences between families (p < 0.05) were detected.

Evaluation of the 1-methyl-2-phenylindole colorimetric assay for aldehydic lipid peroxidation products in plants: malondialdehyde and 4-hydroxynonenal.

The 1-methyl-2-phenylindole colorimetric assay is considered specific for malondialdehyde (MDA) and 4-hydroxynonenal (HNE) in mammalian systems, but its specificity in plant tissues is unknown. This study demonstrates that the assay produces a purple/blue chromophore with an absorbance peak at 586 nm for a malondialdehyde standard, while aqueous extractions from Ribes spp. Beta vulgaris, and Lycopersicon esculentum tissues produce an orange chromophore with an absorbance maximum at 450 nm and a large shoulder that extends to 700 nm. No distinctive MDA peak was discernable in plant samples at lambda=586 nm and absorbance was attributed to background interference. The reaction between sucrose and 1-methyl-2-phenylindole produced an orange chromophore with a spectrum similar to those obtained from plant extractions, suggesting that simple sugars are the likely source of background interference. This study demonstrates that the 1-methyl-2-phenylindole colorimetric assay is non-specific for detecting MDA and HNE in plants and its use is cautioned due to interference, particularly from sugars.

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy.

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).

HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.

Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine.

Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (*OH), utilising DMSO as a radical trap, was used to determine *OH formation during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic Lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (*OH) formation correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation.