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PubMed2005

Survival curves for microbial species stored by freeze-drying.

The survival of a variety of species of microorganism following storage for up to 20 years has been analyzed. The organisms were freeze-dried, sealed in ampoules under vacuum (<1 Pa) and stored in the dark at 5 degrees C. The yeast that was tested, Saccharomyces cerevisiae, showed only 8% survival when recovered shortly after freeze-drying, but subsequent loss during storage was the least among all the tested microorganisms. The decrease in the logarithm of survival per year (log survival) was -0.010, which corresponds to a survival rate of 97.7% per year. The Gram-negative bacteria tested, Escherichia coli, Pseudomonas putida, and Enterobacter cloacae, showed 42.6, 33.5, and 50.8% survival shortly after freeze-drying, which was higher than the corresponding survival of S. cerevisiae, but the subsequent loss during storage was greater than S. cerevisiae, the log survival figures being -0.041, -0.058, and -0.073 per year. These values correspond to survival rates of 91.0, 87.5, and 84.5% each year. The Gram-positive bacteria tested, Lactobacillus acidophilus and Enteroccoccus faecium, showed 62.5 and 85.2% survival shortly after freeze-drying, which was even higher than that of the Gram-negative species, and these organisms also showed better survival during storage than Gram-negative bacteria; their log survival rates were -0.018 and -0.016 per year, which corresponded to survival rates of almost 96% per year. Comparison of these results with other published data for different drying conditions suggests that survival during storage is strongly influenced by the degree of vacuum under which the ampoules were sealed. The excellent survival after freeze-drying of each species might be attributable to the high level of desiccation and to sealing under vacuum.

Freeze Drying↗
PubMed2006

Stability of protein-encapsulating DRV liposomes after freeze-drying: A study with BSA and t-PA.

Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate. Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid = more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.

Animals↗
PubMed1980

The use of freeze-dried blood in the membrane feeding of tsetse flies (Glossina p. palpalis, Diptera: Glossinidae).

Rearing of tsetse flies using membrane feeding became a method of routine during recent years. HOwever, the use of blood does require donor animals. Now, for the first time, a technique has been developed using freeze-dried blood as nutrient for Glossina p. palpalis. This report contains an account of the experiments carried out in obtaining a freeze-dried blood product. Initially, fresh bovine blood was processed prior to freeze-drying. It was later reconstituted with distilled water and the osmolarity adjusted to that of fresh blood. ATP was necessary for optimal blood uptake and the addition of albumin increased the weights of puparia. A colony of flies was reared for more than two years by feeding the flies with this reconstituted blood formulation. Later, in a modified freeze-drying programme the processing of the blood prior to drying was eliminated. The addition of ATP was still required, but the quality of the product was not improved anymore by enrichment with albumin, plasma or serum. In comparisons between freeze-dried blood, fresh blood and in vivo feeding of tsetse flies, no differences were found, except the puparial weights were considerably higher in the colony fed on freeze-dried blood than those of the fresh blood colony. This membrane feeding technique eliminates the dependence on blood donor animals for rearing of tsetse flies and can be recommended for breeding colonies in Africa.

Adenosine Triphosphate↗
PubMed1987

Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue.

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.

Animals↗
PubMed2002

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.

Biosensing Techniques↗
PubMed1989

Freeze-dried bone in pulpotomy procedures in monkey.

The purpose of this study was to evaluate the effect of freeze-dried bone on amputated pulps. Fifteen primary and one permanent monkey teeth were treated with freeze-dried bone. As the control group, another fifteen primary teeth were treated with calcium hydroxide and fifteen primary and one permanent teeth with formocresol. Four other primary teeth pulps were amputated and sealed with tin foil and IRM as controls. Twelve mandibular incisors were left intact. Histologically all but three teeth treated with human freeze-dried bone after three months showed a complete or partial calcific barrier directly below the amputation site. Normal appearing odontoblastic cells were noted below the calcific barrier. The apical third was vital with an occasional chronic inflammatory cell visible. The histological findings of teeth treated with calcium-hydroxide were very similar to freeze-dried bone. All but four teeth showed a complete calcific barrier at the amputation site. The odontoblastic cells were normal in appearance and the inflammatory cell reactions decreased from the middle portion toward the apical region. The histological evaluation of the formocresol-treated teeth was comparable with previously published studies.

Animals↗
PubMed2002

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection.

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 microm PuroSpher RP-18E cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50-2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.

Animals↗
PubMed2004

Paclitaxel antibody conjugates and trehalose for preserving the immunological activity after freeze-drying.

Antibodies and anticancer drug-antibody conjugates used in experimental cancer research or clinically must be freeze-dried for preserving the activity and storage at room temperature. This often results in some denaturation and loss of activity. We describe a recovery of the cytotoxic activity of a paclitaxel-mAb immunoconjugate after freeze-drying. The paclitaxel-antibody conjugate specific for ovarian cancer was tested both for its cytotoxicity in vitro and immunological activity after freeze drying in the presence of various preservatives. Results show that the inclusion of trehalose as a stabilizer at concentrations varying from the 0.25 and 0.40 M protected the antibody and saved the pharmacological activity. When PEG alone or with trehalose was used, the immunological and cytotoxic activities recovery were lower. Albumin was not protective. This study shows that the addition of trehalose for freeze drying labile drug is a promising method for storage of large quantities of the immunoconjugates for experimental and therapeutic use.

Antibodies, Monoclonal↗
PubMed1983

Demonstration of light chain monotypia in B cell non-Hodgkin's lymphomas using unfixed freeze-dried and formalin-fixed trypsinised paraffin sections.

Immunohistological light chain analysis was carried out in 55 patients with non-Hodgkin's lymphoma of B cell origin. Unfixed freeze-dried paraffin sections were used to detect surface Ig and formaldehyde-fixed trypsinised sections to detect cytoplasmic Ig. Cytoplasmic Ig was seen inconstantly in freeze-dried paraffin sections. There was complete agreement with regard to the type of light chain between freeze-dried and formaldehyde-fixed trypsinised sections. Immunohistology showed the monotypic immunoglobulin light chains in 83% of cases when unfixed freeze-dried paraffin sections were used for sIg demonstration while only in 45% of cases when formaldehyde-fixed material was used for cIg detection. The efficiency of sIg demonstration in unfixed freeze-dried paraffin sections was comparable with that based on unfixed cryostat sections or on cell suspensions when the lymphocytic lymphoma/CLL group was excluded from the evaluation. The relatively low frequency of monotypic sIg positivity obtained in this group (12/19) is due to the low density of surface Ig in CLL lymphocytes.

B-Lymphocytes↗
PubMed2002

Preparation and characterization of sterile and freeze-dried sub-200 nm nanoparticles.

The feasibility of producing sterile and freeze-dried polyester nanoparticles was investigated. Various poly(D,L-lactide-co-glycolide) and poly(D,L-lactide) were selected as biodegradable polymers. Using the salting-out procedure, process parameters were optimized to obtain sub-200 nm particles. After purification, the nanoparticle suspensions containing different lyoprotectants were sterilized by filtration. Freeze-drying was performed using vials covered with 0.22 microm membrane filters in order to preserve the suspensions from bacterial contamination. Sterility was assessed on the final product according to pharmacopoeial requirements using the membrane filtration method. With all polymers tested, sub-200 nm particles could be obtained. Nanoparticles with a size as low as 102 nm were prepared with good reproducibility and narrow size distribution. Upon freeze-drying, it appeared that complete redispersion of all types of polyester nanoparticles could be obtained in presence of the lyoprotectants tested such as saccharides while aggregation was observed without lyoprotectant. Sterility testing showed no microbial contamination indicating that sterile nanoparticulate formulations have been achieved.

Capsules↗
PubMed2001

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake.

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.

Animals↗
PubMed1989

Effect of freeze-drying on measurements of pH in biopsy samples of the middle gluteal muscle of the horse: comparison of muscle pH to the pyruvate and lactate content.

Muscle biopsies taken after exercise, in comparison to those at rest, contain increased amounts of blood and this is a particular problem in studies of the horse. The inclusion of blood in muscle will introduce an upward bias in values of pH measured in muscle homogenates. In an attempt to control this, muscle biopsy samples of the middle gluteal from Thoroughbred horses were freeze-dried and dissected free of blood before determination of pH. Following exercise, muscle pH measured after freeze-drying was similar to that measured in homogenates prepared from frozen samples. In contrast, freeze-drying appeared to increase muscle pH in samples taken at rest. This was probably the result of loss of CO2 during freeze-drying. Muscle pH determined in freeze-dried samples taken at rest and after exercise was linearly related to pyruvate and lactate content (P less than 0.001). It is concluded that muscle samples taken after exercise can be freeze-dried and dissected free from blood before determination of pH, whereas this procedure will cause an alkaline shift in samples taken at rest.

Animals↗
PubMed1994

Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying.

The stabilizing effect of mannitol during the freeze-drying of proteins was studied using L-lactate dehydrogenase (LDH, rabbit muscle), beta-galactosidase (Escherichia coli) and L-asparaginase (Erwinia chrysanthemi) as model proteins. Crystallization of mannitol was studied by powder X-ray diffraction and differential scanning calorimetry (DSC), in relation to the stabilizing effect. All the enzymes were protected concentration-dependently by amorphous mannitol, but the stabilizing effect was decreased with an increase in mannitol crystallinity. The heat-treatment of frozen solutions above crystallization temperature prior to drying enhanced mannitol crystallization and LDH inactivation. The importance of maintaining excipients in an amorphous state during freeze-drying, previously reported for Aspergillus oryzae beta-galactosidase (K. Izutsu et al., Pharm. Res., 10, 1233 (1993)), was confirmed using three different enzymes.

Animals↗
PubMed1994

An improved microscope stage for direct observation of freezing and freeze drying.

A microscope stage for observation of freezing and freeze drying is described. The stage uses thermoelectric (Peltier) heaters configured in two stages, with circulating fluid as a heat sink on the high temperature side. Lowest attainable sample temperature is about -47 degrees C. Principal advantages of this system are closed-loop control of stage temperature, rapid response to changes in temperature set point, and improved documentation of experiments by use of a video recorder system with a character generator which allows display of sample identity and temperature. Accuracy of measuring the sample temperature in the field of view was validated by comparing observed values of eutectic melting with published values for a series of solutes with eutectic temperatures in the range from -2 degrees C to -32 degrees C. Good agreement was obtained throughout this range.

Freeze Drying↗
PubMed1995

Leish-KIT, a stable direct agglutination test based on freeze-dried antigen for serodiagnosis of visceral leishmaniasis.

In order to increase the application potential of the direct agglutination test (DAT) for the detection of anti-Leishmania antibodies in human serum samples, we developed an antigen based on stained and freeze-dried Leishmania donovani promastigotes. We describe here the evaluation of the performance of the DAT based on this freeze-dried antigen. It was shown that the freeze-dried antigen remains fully active, even after storage at 56 degrees C for 18 months. With a cutoff value of 1:1,600, the sensitivity of the DAT was shown to be 92% and the specificity of the test was 99.7%, which were comparable with the results found for the DAT based on liquid antigen. The major advantages of the freeze-dried antigen are that the production of a large batch of this antigen allows reproducible results in the DAT over a long period of time and that the freeze-dried antigen can be stored at ambient temperature, which, as was shown, makes the test a valuable diagnostic tool for use in the field.

Agglutination Tests↗
PubMed1992

Study of lipid changes in freeze-dried fish during storage. I. The interaction of relative humidity and tissue lipids.

Several studies on lipid stability of freeze-dried fish pointed out the relationship between relative humidity (RH) and autoxidation of highly unsaturated fatty acids. The present study shows the changes of tissue lipids in two different fish, freeze-dried and stored under various RH conditions. Fillets of Scomber scomber L and Sardina pilchardus sardina were freeze-dried and stored for 1, 3, 6 and 9 months. periodically, the sample of both fish were analyzed to evaluate the autoxidation of lipids. The results show that the autoxidation of lipids is quicker during 1st-3rd month of storage, while in a high humidity environment the tissue lipids change slower. According to these results both time of storage and relative humidity must be controlled to maintain the nutritive value of freeze-dried fish.

Animals↗
PubMedDate not supplied

A biomechanical analysis of solvent-dehydrated and freeze-dried human fascia lata allografts. A preliminary report.

UNLABELLED: This study compares the basic mechanical properties of two groups of commercially available fascia lata allografts processed by different means (solvent-dehydrated and sterilized via gamma radiation, and freeze-dried without secondary sterilization). The results reveal significantly (P less than 0.05) higher stiffness, higher maximum load to failure, and higher maximum load per unit width of graft with the solvent-dried as opposed to the freeze-dried fascia lata. Subsections of individual solvent-dried specimens were also more uniform in their mechanical properties than those of the freeze-dried allografts. CLINICAL RELEVANCE: Fascia lata is used as a graft material in a variety of orthopaedic procedures. Allograft fascia lata offers an increased cross-sectional area of material and eliminates the morbidity associated with the harvesting of autologous tissues. However, the structural uniformity of such large grafts has been questioned. Processing techniques used in the sterilization and storage of such grafts is varied and represents a potential source of variation in the mechanical properties of allograft specimens. The results of this study suggest that a commercially available solvent-dehydrated form of fascia lata provides a more suitable grafting material than freeze-dried specimens obtained from tissue banks.

Adult↗
PubMed1986

The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying.

The fusion of small unilamellar vesicles of phosphatidylcholines during freeze-thawing and freeze-drying/rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide/phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding.

Calorimetry, Differential Scanning↗