Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.     

Appraising freeze-drying for storage of bacteria and their ready access in a rapid toxicity assessment assay

Direct toxicity assessment (DTA) techniques seek to measure the impact of toxic chemicals on biological materials resident in the environment. This study features the use of freeze-dried bacterial cells in combination with a rapid DTA analyser, SciTOX^?. The effects of three factors—cryoprotectant type, bacterial strain, and storage temperature—were tested in order to validate the shelf life of the freeze-dried cells. Three freeze-dried Gram-negative bacterial strains, Acinetobacter calcoaceticus, Escherichia coli and Pseudomonas putida, were tested by using the bacteria in the SciTox^? DTA assay and recording their responses to two standard toxicants: 2,4-dicholorophenol and 3,5-dichlorophenol. Each freeze-dried strain of bacteria was prepared in two forms—either pre-treatment with polyethylene glycol (PEG) or with sucrose/Tween 80—prior to storing at either 4 or ?20 °C for three different storage periods (1, 2 or 3 months). While the sucrose/Tween 80 pre-treated freeze-dried cells exhibited better cell viability, we concluded that PEG was a more suitable cryoprotectant for the bacteria used in the DTA assay because of EC₅₀ parity with fresh cell and zero-time freeze-dried cell assays. The results showed that freeze-dried cells, with appropriate materials and conditions, can give reproducible DTA results for up to 3 months. The availability of a biocomponent that can be activated by simple rehydration makes the deployment of this technology much easier for an end user.     

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.     

Characterization of Injury Incurred by Escherichia coli upon Freeze-Drying

When cells of Escherichia coli ML30 were suspended in 2% gelatin and frozen at -40 C, no appreciable metabolic damage or death occurred. After freeze-drying for 8 hr at a platen temperature of 49 C and rehydration with a mineral salts medium, survival of the cells was 0.6%. Metabolic damage of the survivors was found to be 23%. Permeability alterations were detected by several criteria. Freeze-dried cells were susceptible to antibiotics normally ineffective against E. coli and leakage of ribonucleic acid (RNA) occurred. Analysis of ribosomal extracts of rehydrated freeze-dried cells demonstrated the presence of appreciable degradation products. Permeability alterations were shown to be reversible by the observation that antibiotic susceptibility was a time-dependent process and that the gratuitous inducer of beta-galactosidase was not concentrated by freeze-dried cells until the injured cells had been incubated in a nutrient medium for 300 min or more. At approximately the same time, metabolic damage was repaired. RNA synthesis preceded protein synthesis by about 150 min, and deoxyribonucleic acid synthesis occurred with the resumption of normal growth. This was interpreted to be the result of repair of RNA taking place before protein synthesis and growth could resume. A pronounced increase in the lag time of freeze-dried cells was also observed. Peptides and Casamino Acids shortened the lag time for freeze-dried cells but not for the controls. Glycerol and glucose were found to be better carbon sources for growth of freeze-dried cells than sodium lactate or sodium succinate.     

Effect of package and storage conditions on viability and efficacy of the freeze-dried biocontrol agent Pantoea agglomerans strain CPA-2

AIMS: To reduce concentrations of protective and rehydrating media and to evaluate the effect of storage temperature, packaging and atmosphere conditions on the stability of freeze-dried Pantoea agglomerans cells. Efficacy against Penicillium digitatum of freeze-dried cells in orange fruits was also evaluated. METHODS AND RESULTS: Several concentrations of protective and rehydration media were tested to reduce processing costs. Freeze-dried cells were packed in glass vials or plastic bags under vacuum or nitrogen conditions at 4 and 25 degrees C. After 1 and 3 months, efficacy of freeze-dried P. agglomerans against P. digitatum was tested. CONCLUSIONS: The results indicate that it is possible to reduce the concentration of non-fat skimmed milk as a rehydration medium from 10% to 1%, maintaining viabilities of 100%. Moreover, freeze-dried cells could be stored in glass vials or in high barrier plastic bags at 4 degrees C for 3 months while maintaining high viabilities and efficacy against P. digitatum. SIGNIFICANCE AND IMPACT OF THE STUDY: The major obstacle in the commercialization of biocontrol products is the development of a shelf-stable formulated product that retains biocontrol activity at a level similar to that of fresh cells. This study suggests that it is possible to maintain viability and efficacy of freeze-dried P. agglomerans cells for at least 3 months.     

Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy

Summary Cultured HeLa cells or mouse liver and pancreas tissues were labeled with ³H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes.After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and drymounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.The outline of this paper was presented at the Symposium on Radioautography at the 5th International Congress of Histochemistry and Cytochemistry held in Bucharest, Romania, 30 August–2 September 1976     

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.     

Immunisation of Sheep with Bovine Viral Diarrhoea Virus,E2 Protein Using a Freeze-Dried Hollow Silica Mesoporous Nanoparticle Formulation

Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 μg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 μg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213–500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.     

Preservation by Freeze-Drying and the Stability of Virulence of Salmonella typhimurium

Differences in the ability to withstand freeze-drying were demonstrated among strains of Salmonella typhimurium. On the average, the number of viable cells in freeze-dried cultures stored at 5 C for 12 to 18 months was approximately one half as large as that found 24 hr after freeze-drying. The viability in samples stored at higher temperatures declined rapidly and was correlated with the dryness of the sample. The virulence for mice of three strains of S. typhimurium did not change appreciably when samples were kept for 1 or 2 years as freeze-dried samples stored at 5 C, or as agar cultures stored at 5 C or at room temperature.     

Freeze-Drying of Foot-and-Mouth Disease Virus and Storage Stability of the Infectivity of Dried Virus at 4 C

Foot-and-mouth disease virus, type A, strain 119, propagated in cultures of calf kidney cells and in the tongue epithelium of cattle was used. The process of freeze-drying was conducted in two cycles on unit volumes of 4 ml in Pyrex ampoules, averaging 150 ampoules per run, and was studied separately from the problems of storage. Ampoules containing freeze-dried virus were flame-sealed for either immediate study or storage at 4 C for later reference. Tissue-culture virus dried with various additives had a mean processing loss of 0.8 log LD₅₀ per ml for six different preparations. Virus freeze-dried in tissue suspension had a mean loss of 0.8 log LD₅₀ per ml for three different preparations. A second set of preparations was processed and specifically studied for storage quality at 4 C. The virus in 14 freeze-dried tissue-culture preparations had a mean loss of 0.75 log LD₅₀ per ml while stored at 4 C for 1 year. Virus in four freeze-dried tissue suspensions had a mean loss of 0.05 log LD₅₀ per ml held at 4 C for 1 year. None of the specific additives used for conservation of the virus during the freeze-drying process or during storage at 4 C contributed significantly to the stability of the virus preparations over and above that observed with the normal growth medium of the tissue culture or the ordinary diluents used in making suspensions of tissue virus.     

Proceedings: Factors affecting repair of sublethal injury in frozen or freeze-dried bacteria

Conditions facilitating recovery of sublethally injured cells present in frozen or freeze-dried preparations of bacteria were investigated. Some cells of Streptococcus faecalis injured by freezing required RNA resynthesis for recovery, whereas the rest did not. Addition of 6% NaCl to control recovery medium was found to inhibit RNA synthesis completely in frozen-injured cells of S. faecalis, but not in intact ones. This seems to be a principal reason why 6% NaCl inhibits the growth of the injured cells.The recovery of viable cells of freeze-dried and stored Streptococcus thermophilus was greately increased by the addition of cysteine to the plating medium. The favorable effect of cystein was mainly due to its reducing activity. On the contrary, the addition to the plating medium of certain types of peptone, such as lactalbumin hydrolyzate, resulted in a considerable decrease of viability in frozen preparations of Vibrio metschnikovii.Thus, at present, it seems too early to try to discuss specifically the optimal composition of plating media to give the highest viability from frozen or freeze-dried preparations of bacteria.     

Volatile retention during freeze drying of protein solutions

Retention of ¹⁴C-labeled 2-propanol was studied in freeze-dried systems containing either bovine serum albumin (BSA) or pepsin. Retention during freeze drying varied with: initial concentration of solids and 2-propanol, rate of freezing and sample thickness. Heat denaturation of the proteins did not significantly affect the volatile retention. When freeze-dried BSA was rehumidified, volatile retention dropped to a new level which depended on the final water content. Heating the freeze-dried BSA systems in vacuum had no effect on the 2-propanol retention. The results are indicative of structurally dependent retention and can be mainly interpreted by the “microregions” theory of volatile retention.     

Enhancement of Flowering by Acetic Acid Bacteria-oxidized Products in Pharbitis Seedlings

When seedlings of Pharbitis nil Chois., strain Shifukurin weregrown in a medium containing acetic acid bacteria-oxidized ethanol(AABOE) or acetic acid bacteria-oxidized methanol (AABOM), 100%flowering was induced by exposure to a single dark period of9.5 to 10.5 hr, while this regime induced little or no floweringin control plants. When the plants were grown in water containingthe freeze-dried substance from an AABOE solution, floweringwas also enhanced. Vegetative growth was considerably suppressedby these fermented alcohols and by the freeze-dried substancefrom an AABOE solution. However, the freeze-dried substancefrom the AABOM solution had no effect on either flowering orvegetative growth. (Received May 13, 1981; Accepted August 21, 1981)     

Studies on the capacity of B cells as well as T cells to serve as accessory cells for the activation of herpes simplex virus-specific cytolytic T cells

Experiments were conducted in an effort to determine the ability of B and T lymphocytes to serve as APC for the activation of HSV-primed splenic T cells to become class I-restricted, HSV-specific CTL. The results showed that both freshly isolated splenic B cells as well as LPS and dextran sulfate (L/D)-activated B cells were effective at stimulating the generation of CTL during a 5-day in vitro culture. There was no requirement for the addition of exogenous IL-2 to the culture and, since murine B cells do not appear to express either membrane or secreted IL-1, this lymphokine appears to either not be required for the activation of virus-specific CTL or to be provided by the T cells themselves. When normal B cells were separated into fractions enriched for resting vs activated cells and then tested for their ability to stimulate the generation of HSV-specific CTL, it was found that while the activated B cells were quite effective at stimulating the generation of CTL, resting B cells were ineffective at carrying out this function. In contrast to normal B cells, normal T cells were unable to act as APC. However, Con A-activated T lymphoblasts were equivalent to L/D B cells in their ability to mediate the generation of CTL activity. L/D B cells that had been pulsed with HSV and then incubated at 37 degrees C for greater than 1 h could be fixed with paraformaldehyde and were still able to function as APC. The finding that L/D B cells, that had been fixed at 1 h or less after exposure to HSV, were unable to function as APC suggested that either active Ag "processing" steps may be required for the presentation of Ag in the context of class I molecules or that there is a requirement for the synthesis of viral protein Ag before presentation.     

Binding of concanavalin-A to critical-point-dried and freeze-dried human lymphocytes

When human lymphocytes are treated with concanavalin-A (con A) and hemocyanin, the hemocyanin marker, which demonstrates con A binding sites, can be visualized by scanning (SEM) and transmission electron microscopy (TEM) on both critical-point-dried and freeze-dried cells. The ability to visualize the hemocyanin marker by SEM, its quantity and distribution, were all similar in lymphocytes prepared by both drying procedures. By TEM, hemocyanin was seen adjacent to the plasma membrane on critical-point-dried lymphocytes. In contrast, freeze-dried cells showed hemocyanin labeling at some distance from the plasma membrane (40-70 nm) as well as adjacent to it. The distribution of hemocyanin corresponded to the thickness of the amorphous coat seen on fixed, freeze-dried cells. Therefore, the extracellular coat on freeze-dried lymphocytes is a carbohydrate-containing glycocalyx.     

FREEZE-DRYING OF BLUE-GREEN ALGAE FOR ELECTRON MICROSCOPE STUDIES1

Cultures of Anabaena cylindrica Lemm. and Eucapsis sp. were harvested, freeze-dried, and prepared for electron microscope studies. A comparison of the freeze-dried preparations was made with control specimens not freeze-dried. Fixation and embedding were according to the Ryter and Kellenberger technique. The photosynthelic thylakoids of the freeze-dried cells were more distinct and less distorted than those of the control cells. Freeze-dried Eucapsis sp. cells exhibited a prominent mucous capsule, whereas in the nonfreeze-dried, no such capsule could be discerned.     

Alternative pathways of glucose transport in Prevotella bryantii B(1)4(1)

Prevotella bryantii B(1)4 grew faster on glucose than mannose (0.70 versus 0.45 h(-1)), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both (14)C-glucose and (14)C-mannose, but cells grown on glucose had over three-fold higher rates of (14)C-glucose transport than cells grown on mannose. The (14)C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower (14)C-glucose transport, but the (14)C-mannose transport did not change significantly. (14)C-glucose and (14)C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The (14)C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (K(i)=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B(1)4 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.     

Preservation of tissue mucins by freeze-drying and vapour fixation

Summary Histochemical studies previously undertaken showed that tissue mucins (glycoproteins) of the rat submaxillary salivary gland and other organs are preserved very satisfactorily by formaldehyde vapour treatment applied after freeze-drying of the tissue.It was thought desirable to confirm these histochemical findings by quantitative chemical data. This was performed by studying the effect of formaldehyde vapour treatment on the solubility of proteins in the freeze-dried rat submaxillary gland.Large quantities of protein (about 30 to 60 per cent of the dry weight) could be removed by aqueous extraction from the freeze-dried control samples, which had not received any formaldehyde vapour treatment, but very little protein (about 0.5 to 4 per cent of the dry weight) could be extracted from those samples which had been exposed to this vapour at 50°C for 3 hours.Each of the experiments performed confirmed this overall picture, but there were differences in the amount of protein extracted among the control samples, as well as among the formaldehyde vapour treated ones; it has been suggested that these differences were due to variations in the proportion and/or type of protein present, most probably caused by fluctuations in the content of secretory mucins.In part fulfilment for the Doctorate of Philosophy, University of London.     

Production of concentrated freeze-dried cultures of Bifidobacterium longumin k-carrageenan-locust bean gum gel

Bifidobacterium longum was immobilized in k-carrageenan/locust bean gum gel beads, and cultured in a medium containing Lactobacillus MRS broth and whey-permeate. The same beads were incubated for 5 successive batch fermentations and freeze-dried following mixing with a protective solution. Viable population in the beads increased from 8 3 10 7 to 4.7 3 10 10 cfu/g after three batch fermentations, but no further increase in viable cell population could be achieved in the last two fermentations. The freeze-dried culture contained 3 3 10 10 cfu/g with a survival rate of approximately 10%. Survival to freeze-drying of immobilized cells was as good as that of classical free-cell cultures. Stability of freeze-dried cultures during storage at minus 17, 4 and 20°C was not influenced by immobilization.     

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.