Simplified cryopreservation of the microalga Chlorella vulgaris integrating a novel concept for cell viability estimation

Microalgae currently receive growing attention as promising candidates for future bio‐economy concepts. However, the reliable maintenance of production strains remains challenging. The well‐established serial subculturing techniques suffer from low long‐time stability and high effort and are therefore stepwise being replaced by cryopreservation. Currently, available protocols are often deduced from cell culture technology and are rather complex. This study aimed to investigate if less complex approaches can be applied. We introduce an easy‐to‐use cryopreservation protocol based on the model organism Chlorella vulgaris. To overcome error‐prone viability estimation by plating techniques, an alternative method using growth pattern analysis was developed. As revealed by growth pattern analysis, the preservation of stationary phase cells proved superior to the commonly applied concept of freezing cells from the growing phase. Controlled‐rate cooling using simple devices resulted in reproducibly high post‐thawing viabilities in the range of 63 ± 2%. Moreover, the presented protocol highlights the potential of simplifying microalgal cryo‐preservation procedures, thereby reducing the required labor and material need to a minimum. Apart from the viability analysis of the cryopreserved microalga C. vulgaris, this approach seems to have the potential to be applied for other algae species and microorganisms, as well.     

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho‐associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow‐freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post‐thaw culture, the presence of 10 μM ROCK inhibitor (Y‐27632) and 1 μM pifithrin‐μ together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder‐dependent or feeder‐independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A simple and efficient method for cryopreservation of embryogenic triticale calli

Conventional breeding methods are now supplemented by modern in vitro techniques. However, long term maintenance of cultures has many disadvantages. It incurs risk of loss through microbial contamination, somaclonal variation or human error, but above all the regeneration capacity can decrease gradually during extended maintenance. Cryopreservation as a method of long term storage of biological material without genetic alteration was adapted for embryogenic triticale calli preservation. Callus of both winter and spring genotypes were successfully cryopreserved. The best viability rates (80–85%) were achieved with 6 weeks old winter genotypes treated with cryoprotective solution containing DMSO. This simple and efficient method of cryopreservation requires no special devices for controlled freezing and can be easily adapted for other cereals.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.     

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.     

A comparison between freezing methods for the cryopreservation of stallion spermatozoa

The effects of sperm freezing concentration (40 × 10⁶ mL⁻¹ vs. 400 × 10⁶ mL⁻¹), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam^® box vs. programmable freezing machine) were evaluated in a 2 × 2 × 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 × 10⁶ mL⁻¹ was higher than for spermatozoa frozen at 400 × 10⁶ mL⁻¹. No significant differences were observed in the semen parameters assessed after cryopreservation in either 0.25 or 0.5 mL straws. However, the programmable freezer provided a more consistent and reliable freezing rate than liquid nitrogen vapour. We conclude that an effective protocol for the cryopreservation of stallion spermatozoa at low concentrations would include concentrations of 40 × 10⁶ mL⁻¹ in 0.25 mL straws using a programmable freezer. This freezing protocol would be suitable for emerging sperm technologies such as sex-preselection of stallion spermatozoa as the sorting process yields only low numbers of spermatozoa in a small volume available for either immediate insemination or cryopreservation.     

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes

Strict anaerobic gut microbes have been suggested as ‘next‐generation probiotics’ for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (?80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant‐free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant‐free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process‐ and species‐specific.     

Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)

Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube.     

Cryopreservation of <Emphasis Type="Italic">Panax ginseng</Emphasis> Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L⁻¹ was recorded for post-freeze-regenerated cultures versus 9.1 g dw L⁻¹ for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.     

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, −1 °C/min chilling rate (18 °C to 5 °C) and −13 °C/min freezing rate (5 °C to −80 °C), followed by immersion in liquid nitrogen.     

Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica

Crypopreservation methods were firstly developed for root-tips from hairy root cultures of Beta vulgaris, established after transformation by Agrobacterium rhizogenes. The effects of culture age, pre-growth, cryoprotection, freezing rate and post-freeze culture conditions were determined. The resulting freezing protocol was then used to cryopreserve transformed root cultures of Nicotiana rustica. Both species were viable after freezing (ca. 80%), according to fluorescein diacetate vital staining. However, on average the regeneration of proliferating roots from surviving root-tips was low (<20%). Growth rates, secondary metabolite production and T-DNA structure of a number of hairy root lines were examined and found to be unchanged after cryopreservation.Abbreviations DMSO dimethyl sulphoxide - 2,4-D dichlorophenoxyacetic acid - FDA fluoroscein diacetate - IAA indole-3-acetic acid - NAA -napthalene acetic acid     

Dynamic co‐culture metabolic models reveal the fermentation dynamics,metabolic capacities and interplays of cheese starter cultures

In this study, we have investigated the cheese starter culture as a microbial community through a question: can the metabolic behaviour of a co‐culture be explained by the characterized individual organism that constituted the co‐culture? To address this question, the dairy‐origin lactic acid bacteria Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Leuconostoc mesenteroides, commonly used in cheese starter cultures, were grown in pure and four different co‐cultures. We used a dynamic metabolic modelling approach based on the integration of the genome‐scale metabolic networks of the involved organisms to simulate the co‐cultures. The strain‐specific kinetic parameters of dynamic models were estimated using the pure culture experiments and they were subsequently applied to co‐culture models. Biomass, carbon source, lactic acid and most of the amino acid concentration profiles simulated by the co‐culture models fit closely to the experimental results and the co‐culture models explained the mechanisms behind the dynamic microbial abundance. We then applied the co‐culture models to estimate further information on the co‐cultures that could not be obtained by the experimental method used. This includes estimation of the profile of various metabolites in the co‐culture medium such as flavour compounds produced and the individual organism level metabolic exchange flux profiles, which revealed the potential metabolic interactions between organisms in the co‐cultures.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

Root cryopreservation to biobank medicinal plants: a case study for Hypericum perforatum L.

In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L⁻¹ gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species.

     

Efficient cryopreservation of adventitious shoots of Begonia x erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline

Despite the widespread use of tissue culture as a means of propagating begonias and concerns regarding the preservation of germplasm, little information is available on the cryopreservation of these commercially important plants. For this reason studies were conducted to develop an encapsulation–dehydration method for the cryopreservation of adventitious shoots of the rhizomatous begonia, Begonia x erythrophylla. Adventitious shoots of B. x erythrophylla were found to be sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford cryoprotection without prior dehydration. Addition of abscisic acid and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75 M sucrose, 3.8 μM abscisic acid and 2.15 mM proline resulted in greater than 50% of shoots surviving freezing.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.