Freeze-thaw Technique Research Articles

PBS incubation, a simple technique to release miracidia of Opisthorchis-like eggs for DNA extraction

This study describes a simple technique for releasing miracidia from Opisthorchis-like eggs before DNA extraction by keeping PBS washed specimens at 4 °C for two days. Using PCR amplification, the sensitivity of the PBS incubation technique was as good as that obtained from freeze–thaw technique. Moreover, this low-cost technique is less laborious.

Effects of ergosterol on the structure and activity ofNeurosporamitochondrial porin in liposomes

Mitochondrial porins (also known as voltage-dependent anion-selective channels (VDACs)) regulate and contribute to cellular metabolism. These proteins copurify with sterols, and some purified forms of the protein require sterol for insertion into planar artificial membranes. Recently, interactions between detergent-solubilized mitochondrial porins and sterols have been detected by NMR and spectroscopic methods, but the effects of sterols on pore function remained to be assessed. Therefore, in this work, a freeze-thaw technique was used to introduce recombinant Neurospora porin into liposomes containing, or lacking, the native fungal sterol ergosterol. In both types of liposomes, insertion of the protein converts it to a protease-resistant state and low levels of dimeric and trimeric forms are observed. There are only minor differences between the secondary structural components of the protein in the presence or absence of sterol. Ergosterol in proteoliposomes alters their osmotic responses to sucrose, possibly due to increased membrane rigidity or interactions with the protein that were not revealed by the methods used in this study. The presence of ergosterol is associated with an increased change in conformation and loss of function of liposome-embedded porin at high temperature. Taken with other evidence for direct interactions of sterols with porins, these results support a link between these two molecules in mitochondrial membrane activity.

Thermal behavior and mechanical properties of physically crosslinked PVA/Gelatin hydrogels

Poly (vinyl alcohol)/Gelatin hydrogels are under active investigation as potential vascular cell culture biomaterials, tissue models and vascular implants. The PVA/Gelatin hydrogels are physically crosslinked by the freeze-thaw technique, which is followed by a coagulation bath treatment. In this study, the thermal behavior of the gels was examined by differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). Rheological measurement and uniaxial tensile tests revealed key mechanical properties. The role of polymer fraction in relation to these mechanical properties is explored. Gelatin has no significant effect on the thermal behavior of PVA, which indicates that no substantial change occurs in the PVA crystallite due to the presence of gelatin. The glass transition temperature, melting temperature, degree of crystallinity, polymer fraction, storage modulus ( G ′ ) and ultimate strength of one freeze-thaw cycle (1FT) hydrogels are inferior to those of 3FT hydrogels. With coagulation, both 1FT and 3FT hydrogels shifted to a lower value of T g , melting temperature and polymer fraction are further increased and the degree of crystallinity is depressed. The mechanical properties of 1FT, but not 3FT, were strengthened with coagulation treatment. This study gives a detailed investigation of the microstructure formation of PVA/Gelatin hydrogel in each stage of physical treatments which helps us to explain the role of physical treatments in tuning their physical properties for biomechanical applications.

Stability Through the Ages: The GSK Experience

It is common knowledge in the pharmaceutical industry that the quality of a company's compound collection has a major influence on the success of biological screening in drug discovery programs. DMSO is the widely accepted solvent of choice for storage of compounds, despite the hygroscopic nature of the solvent, which can lead to stability issues. Other factors that can affect compound stability (e.g., degradation, precipitation) include concentration of compound, intrinsic compound stability, presence of reactive contaminants, storage format-related factors (vessel, sealing, etc.), storage conditions (temperature, humidity, freeze-thaw technique and cycles, etc.), and storage time. To define the best practice for the storage and handling of solution samples, GlaxoSmithKline has undertaken stability experiments over more than a decade, initially to support the implementation of new automated liquid stores (ALS) and, subsequently, to enhance storage and use of compounds in solution through an understanding of compound degradation under storage and assay conditions. The experiments described used a number of technologies, including hyphenated liquid chromatography, electrospray mass spectrometry, flow chemiluminescence nitrogen detection, nuclear magnetic resonance, and Karl Fischer titration.

Physically Cross-Linked Polyvinyl Alcohol for the Topical Delivery of Fluconazole

The aim of this study was to develop fluconazole in an ultrapure polyvinyl alcohol (PVA) hydrogel able to deliver the drug in a sustained release pattern for local treatment of skin fungal infections. The topical fluconazole hydrogels were prepared using PVA hydrogels physically cross-linked by freeze–thaw technique. Polyethylene glycol (PEG) was added as a hydrophilic excipient as a release enhancer of fluconazole. The effects of PVA molecular weight, PEG molecular weight, and PEG concentration were studied using a 2 × 4 × 2 factorially designed experiment. The selected fluconazole hydrogel proved to be physically stable over a period of 6 months and to be effective in the topical treatment of cutaneous candidiasis. Therefore, it could be concluded that the formula composed of 10% PVA 205000 and 1.5% PEG 4000 and 2% fluconazole and prepared by three cycles of freezing, and thawing is very promising in the local treatment of skin fungal infection as an alternative to the systemic use of fluconazole.

Increasing the Entrapment of Protein-Loaded Liposomes with a Modified Freeze–Thaw Technique: A Preliminary Experimental Study

The objective of this study was to investigate the possibility of increasing the entrapment of protein-loaded liposomes with a modified freeze–thaw technique. Blank liposomes were prepared by ethanol injection method. Then recombinant human growth hormone (rhGH) was added to blank liposome suspension. rhGH encapsulation efficiency was enhanced by modified freeze–thaw technique. We separated each step and studied the effect of each parameter on encapsulation: incubation temperature (water bath from 0 to 15°C), incubation duration (from 5 to 90 min), number of freeze–thaw (from 0 to 4 cycle), and the ratio between rhGH and phospholipids (from 1:5 to 1:10). The effect of cryoprotectants on the encapsulation and particle size distribution was finally examined. rhGH encapsulation efficiency was determined by Bradford's dye-binding assay. Morphology and size distribution of rhGH liposomes were also observed. The optimum parameters for rhGH encapsulation were incubation temperature of 5°C, incubation duration of 40 min, and three to four cycles of freeze–thaw. The ratio between rhGH and phospholipids did not affect the encapsulation percentage. Trehalose exhibited the highest integrated value among the cryoprotectants investigated. From the results, this study demonstrates the suitability of the modified freeze–thaw technique for obtaining rhGH liposomes with high entrapment.

Hemolysis falsely decreases intraoperative parathyroid hormone levels

Background Intraoperative parathyroid hormone (IOPTH) measurement is used to confirm biochemical cure during parathyroidectomy. Falsely decreased IOPTH measurements could result in false-negative or false-positive results and lead to failed parathyroidectomy or unnecessary additional exploration. Study design The records of all patients who underwent parathyroidectomy with IOPTH between May and August 2007 were retrospectively reviewed, and the frequency of hemolysis of IOPTH samples was determined. Separately, 10 split-samples were hemolyzed using the freeze-thaw technique. Results Forty-seven patients underwent parathyroidectomy, and 226 IOPTH samples were sent. Seventeen (7.5%) specimens from 9 (18.8%) patients were hemolyzed. In 8 split-samples, the range of decrease caused by hemolysis was 24.5% to 53.8% compared with nonhemolyzed controls. Conclusions Hemolysis of IOPTH samples occurs commonly and falsely decreases IOPTH levels. Unrecognized hemolysis in pre-excision specimens could result in false-negative IOPTH results and lead to unnecessary continued exploration. Unrecognized hemolysis in postexcision specimens could lead to false-positive IOPTH results and lead to failed parathyroidectomy and the need for reoperation. Thus, hemolysis may be an easily preventable cause of erroneous IOPTH measurements.

Liquid Nitrogen Cryotherapy of Superior Limbic Keratoconjunctivitis

To evaluate the effects of liquid nitrogen cryotherapy on superior limbic keratoconjunctivitis (SLK). Interventional case series. In this clinical practice case series, the effects of liquid nitrogen cryotherapy on SLK were observed. Liquid nitrogen cryotherapy was performed using a Brymill E tip spray (0.013-inch aperture) with a double freeze-thaw technique. All subjects were outpatients who had local anesthesia with a single drop of topical proparacaine. The main outcome measure was the resolution of the disease process after treatment. Four female patients (average age, 64 +/- 13 years) and seven eyes with SLK were treated with liquid nitrogen cryotherapy. Resolution of signs and symptoms occurred within two weeks. Disease recurred in two patients and three of seven eyes, although repeat cryotherapy eradicated SLK in all cases. The repeat cryotherapy was performed at three months postoperatively. There were no adverse ocular events. Liquid nitrogen cryotherapy appears to be an effective alternative treatment for SLK as all subjects studied achieved long-term cures. Repeat cryotherapy may be necessary in some instances and may be performed three months after the first treatment.

Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement

Neural progenitor cell grafting is a promising therapeutic option in the treatment of Parkinson's disease. In previous experiments we grafted temperature-sensitive immortalized CSM14.1 cells, derived from the ventral mesencephalon of E14-rats, bilaterally in the caudate putamen of adult hemiparkinsonian rats. In these studies we were not able to demonstrate either a therapeutic improvement or neuronal differentiation of transplanted cells. Here we examined whether CSM14.1 cells grafted bilaterally orthotopically in the substantia nigra of hemiparkinsonian rats have the potential to differentiate into dopaminergic neurons. Adult male rats received 6-hydroxydopamine into the right medial forebrain bundle, and successful lesions were evaluated with apomorphine-induced rotations 12 days after surgery. Two weeks after a successful lesion the animals received bilateral intranigral grafts consisting of either about 50 000 PKH26-labelled undifferentiated CSM14.1 cells (n = 16) or a sham-graft (n = 9). Rotations were evaluated 3, 6, 9 and 12 weeks post-grafting. Animals were finally perfused with 4% paraformaldehyde. Cryoprotected brain slices were prepared for immunohistochemistry using the freeze-thaw technique to preserve PKH26-labelling. Slices were immunostained against neuronal epitopes (NeuN, tyrosine hydroxylase) or glial fibrillary acidic protein. The CSM14.1-cell grafts significantly reduced the apomorphine-induced rotations 12 weeks post-grafting compared to the sham-grafts (P < 0.05). There was an extensive mediolateral migration (400-700 microm) of the PKH26-labelled cells within the host substantia nigra. Colocalization with NeuN or glial fibrillary acidic protein in transplanted cells was confirmed with confocal microscopy. No tyrosine hydroxylase-immunoreactive grafted cells were detectable. The therapeutic effect of the CSM14.1 cells could be explained either by their glial cell-derived neurotrophic factor-expression or their neural differentiation with positive effects on the basal ganglia neuronal networks.

Ethanol Acutely Stimulates Islet Blood Flow, Amplifies Insulin Secretion, and Induces Hypoglycemia via Nitric Oxide and Vagally Mediated Mechanisms

Hypoglycemia induced by alcohol ingestion is a well-known problem in diabetic patients. However, the mechanisms underlying this phenomenon have largely remained elusive. Because insulin secretion in vivo can be rapidly tuned by changes in pancreatic microcirculation, we evaluated the influence of acute alcohol administration on pancreatic islet blood flow (IBF), and dynamic changes in insulin secretion and glycemia in the rat. Ethanol (10%) or saline was iv injected as a bolus into Wistar rats, yielding serum ethanol concentrations of approximately 8 mmol/liter. Measurements of pancreatic blood flow (PBF) were performed by a microsphere technique in combination with a freeze-thawing technique after 10-min injection. Ethanol preferentially and significantly increased pancreatic IBF approximately 4-fold, whereas not influencing whole PBF. The alcohol also augmented late-phase insulin secretion and induced late hypoglycemia upon ip glucose tolerance tests. The nitric oxide synthase inhibitor N-w-nitro-L-arginine methyl ester and atropine prevented the increased pancreatic IBF, enhanced insulin secretion, and hypoglycemia evoked by ethanol. Thus, our findings demonstrate that ethanol acutely exerts substantial influences on pancreatic microcirculation by evoking a massive redistribution of PBF from the exocrine into the endocrine part via mechanisms mediated by nitric oxide and vagal stimuli, augmenting late-phase insulin secretion, and thereby evoking hypoglycemia. This effect may in part underlie the well-known hypoglycemic properties of alcohol in diabetic patients or in alcoholics with hepatic failure.

Transformation of Agrobacterium Using the Freeze-Thaw Method

INTRODUCTIONThis protocol describes a method for transforming Agrobacterium with plasmid DNA using a freeze-thaw technique. Although the transformation efficiency for Agrobacterium is lower than that for Escherichia coli, it is possible to obtain adequate numbers of transformants with this technique.

DNA extraction and PCR detection of Paenibacillus larvae spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P.larvae detection in natural samples.

A permittivity study for PVA membranes, obtained by freeze-thaw technique

Late diffusion or selective diffusion through polymeric membranes has various applications in separation processes, environmental protection, pharmaceutics for drug delivery systems, etc. The aim of this work is to present the results of some ions diffusion through poly(vinyl alcohol) [PVA] hydrogel membranes, obtained by freeze-thaw technique. The diffusion of potentially toxic ions -Pb(II)- and of alkaline ions like Na+ and K+ is tested using conductometry, flame photometry and atomic absorption spectrometry. The membrane permitivitty is calculated for every ionic species and the opportunity is studied to use these membranes in environmental protection, for waste water purification. The modifications of the PVA structure have been made by mixing the PVA solution with some natural products (beta-cyclodextrine or chitosan) or by compounding PVA hydrogel with Cu2+.

Freeze-thaw increases adeno-associated virus transduction of cells

A combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells (128-fold) in comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1,174 +/- 69.8 microM2 per cell. Reduction of cryogenic medium volume and repeated freeze-thaw further increased transduction efficiency by 2.8- and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide, and cold temperatures, which are also involved in cell preservation, had no significant impact on transduction efficiency. Increased transduction was also observed in mesenchymal stem cells (42-fold) by the freeze-thaw method. The potential mechanism of this novel technique likely involves an increase in the net permeable area of biological membranes caused by water crystallization. These findings provide a new approach for gene delivery in various cell types, particularly in those resistant to transduction by conventional methods.

Effect of oxygen on the NMR relaxation properties of crude oils

Nuclear magnetic resonance relaxation measurements of bulk fluids provide a sensitive probe of the dynamics of molecular motion. Dissolved oxygen can interfere with this technique as its paramagnetic nature leads to a reduction of the paramagnetic relaxation times of the fluids. We studied this effect for the relaxation properties of crude oils that are in general characterized by a distribution of relaxation times. The samples were stock tank oils that have been exposed to air. We comparedT1 andT2 relaxation time distributions and their correlation functions of the initial (oxygenated) samples with those from the deoxygenated samples. Oxygen was removed from the oils with a freeze-thaw technique. As expected, the effect of oxygen is most apparent in oils with long relaxation times. In these oils the effect of oxygen can be described by an additional relaxation rate 1/T1,2ox to the transverse and longitudinal relaxation rates that is sample dependent but does not vary within the relaxation time distribution of the oil. Values of 1/T1,2ox for different crude oils were found to be in the range of 2.5 to 8.3 s. For crude oils that have components with relaxation times less than 100 ms, no significant oxygen effect is observed.

Functional activity of insulinoma cells (INS‐1E) and pancreatic islets cultured in agarose cryogel sponges

Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for culturing both isolated pancreatic islets and insulinoma cells (INS-1E). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply.

Effects of Repetitive Vitrification on the Survival of Mouse Oocytes

Cancer survivors often lose their fertility before bearing children due to radiation, chemotherapy, and surgery. It is, therefore, important to develop oocyte cryopreservation techniques to extend their fertility for subsequent childbearing. In this study, we investigated the survival rates of oocytes after a repetitive freeze-thaw technique using vitrification.

Cryopreservation of human embryonic stem cells without the use of a programmable freezer

An effective freezing-thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing-rapid thawing without a programmable freezer. The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved-thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.

373 SPERM TREATMENT AFFECTS THE EFFICIENCY OF PORCINE TRANSGENIC EMBRYO PRODUCTION BY ICSI

Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis, consisting of the use of sperm cells as a vector for transmitting exogenous DNA into eggs at the moment of fertilization. A degradation of sperm membrane followed by incubation with DNA and fertilization using intracytoplasmic sperm injection (ICSI) proved to be efficient in mice (Perry et al. 1999 Science 284, 1180-1183; Moreira et al. 2004 Biol. Reprod. 71, 1943-1947) and rats (Kato et al. 2004 Mol. Reprod. Dev. 69, 153-158). In this study, we evaluated the effect of the sperm treatment (a quick freezing-thawing process for disrupting sperm membranes) on further transgenic expression and the embryo development of injected porcine oocytes. Ejaculated sperm cells from five fertile mature boars were used as vectors for transferring plasmid DNA (GFP: green fluorescent protein) into matured porcine in vitro oocytes by ICSI. Semen was recovered and immediately diluted 1:10 in SFM (swine fertilize medium) at 37�C and later centrifuged (800g, 10 min, 25�C), discarding the seminal plasma to avoid a detrimental effect on DNA binding to cells without further preparation (control), or after they had been subjected to membrane disruption by a quick freeze-thawing process (FT). Linealized plasmid DNA (5.4 kb) was added (1 � 108 sperm/mL + 5 mg DNA/mL) and incubated at 16�C for 30 min. 5 min before ICSI, sperm were pre-warmed at 37�C. Denuded oocytes were washed twice in DPBS medium supplemented with 10% FCS (fetal calf serum) and transferred to ICSI drops. Injected oocytes were kept in TALP medium (Rath et al. 1999 J. Anim. Sci. 77, 3346-3352) for 18 h, and then transferred to NCSU-23 medium for further embryo culture. Embryos were examined for cleavage rate at 48 h following injection, and for embryo development at 144 h. GFP expression in embryos was examined under fluorescent light using a fluorescence inverted microscope. In this preliminary study, 105 and 101 oocytes were injected for control and FT groups, respectively. The cleavage rate was similar between groups (control: 49/105 (46.7%) vs. FT: 39/101 (38.6%); P = 0.25). However, the blastocyst formation rate was lower in the FT than in control group (control: 13/105 (12.4%) vs. FT: 4/101 (4.0%); P = 0.03). In relation to transgenic expression, the FT group showed a significantly higher number of transgenic embryos (control: 50.98 % vs. FT: 75.51%; P = 0.01). These results confirmed that treatment of the sperm prior to the ICSI could affect the efficiency of the production of transgenic embryos. The disruption of sperm membrane increases the DNA binding and the possibilities of carrying the DNA into the oocyte, and freezing-thawing technique is simple and effective to achieve this purpose. However, the freezing process could damage the sperm nucleus structure and decrease the viability of the embryo produced. Nevertheless, the efficiency of the transgenesis expression was very high (more than 75% of the embryos were obtained), and it should be a useful tool to produce transgenic pigs. This work was supported by AGL2003-03144.

Freeze–thaw programmes rescue the implantation of day 6 blastocysts

The developmental rate of a blastocyst is considered one of the main estimates for evaluating the implantation potential of embryos. Day 6 blastocysts have been reported to be much less viable than day 5 blastocysts. Regarding implantation, the implantation window is advanced due to a background of high sex hormones, and slower growing embryos may not implant because of possible desynchrony with the implantation window. The aim of this study was to investigate the efficacy of cryopreservation of such embryos and subsequent synchronization of embryo transfer with endometrial status. The results of 122 day 6 blastocysts transferred in the clinic were retrospectively examined. Pregnancy rates were compared between the stimulation cycle and hormone replacement cycle in terms of the method of endometrial preparation. Fifty-five day 6 blastocysts were transferred onto the stimulation cycle endometrium in 37 women, resulting in a 5.5% viable pregnancy rate. On the other hand, 67 day 6 blastocysts were transferred onto endometrium prepared by exogenous hormones in 40 women, resulting in a 26.9% viable pregnancy rate ( P < 0.01). Consequently, the difference was highly significant. In conclusion, synchronous transfer of slow-growing embryos using the freeze–thaw technique contributes to a positive outcome.