[Effects of freeze-drying bovine pericardium using a combination of polyethylene glycol and trehalose].

The freeze-drying is a technology that preserves biological samples in a dry state, which is beneficial for storage, transportation, and cost saving. In this study, the bovine pericardium was treated with a freeze-drying protectant composed of polyethylene glycol (PEG) and trehalose (Tre), and then freeze-dried. The results demonstrated that the mechanical properties of the pericardium treated with PEG + 10% w/v Tre were superior to those of the pericardium fixed with glutaraldehyde (GA). The wet state water content of the rehydrated pericardium, determined using the Karl Fischer method, was (74.81 ± 1.44)%, which was comparable to that of the GA-fixed pericardium. The dry state water content was significantly reduced to (8.64 ± 1.52)%, indicating effective dehydration during the freeze-drying process. Differential scanning calorimetry (DSC) testing revealed that the thermal shrinkage temperature of the pericardium was (84.96 ± 0.49) ℃, higher than that of the GA-fixed pericardium (83.14 ± 0.11) ℃, indicating greater thermal stability. Fourier transform infrared spectroscopy (FTIR) results showed no damage to the protein structure during freeze-drying. Hematoxylin and eosin (HE) staining demonstrated that the freeze-drying process reduced pore formation, prevented ice crystal growth, and resulted in a tighter arrangement of tissue fibers. The frozen-dried bovine pericardium was subjected to tests for cell viability and hemolysis rate. The results revealed a cell proliferation rate of (77.87 ± 0.49)%, corresponding to a toxicity grade of 1. Additionally, the hemolysis rate was (0.17 ± 0.02)%, which is below the standard of 5%. These findings indicated that the frozen-dried bovine pericardium exhibited satisfactory performance in terms of cytotoxicity and hemolysis, thus meeting the relevant standards. In summary, the performance of the bovine pericardium treated with PEG + 10% w/v Tre and subjected to freeze-drying could meet the required standards.

Nanogels with covalently bound and releasable trehalose for autophagy stimulation in atherosclerosis.

Atherosclerosis, cholesterol-driven plaque formation in arteries, is a complex multicellular disease which is a leading cause of vascular diseases. During the progression of atherosclerosis, the autophagic function is impaired, resulting in lipid accumulation-mediated foam cell formation. The stimulation of autophagy is crucial for the recovery of cellular recycling process. One of the potential autophagy inducers is trehalose, a naturally occurring non-reducing disaccharide. However, trehalose has poor bioavailability due to its hydrophilic nature which results in poor penetration through cell membranes. To enhance its bioavailability, we developed trehalose-releasing nanogels (TNG) for the treatment of atherosclerosis. The nanogels were fabricated through copolymerization of 6-O-acryloyl-trehalose with the selected acrylamide-type monomers affording a high trehalose conjugation (~ 58%, w/w). TNG showed a relatively small hydrodynamic diameter (dH, 67 nm) and a uniform spherical shape and were characterized by negative ζ potential (-18 mV). Thanks to the trehalose-rich content, TNG demonstrated excellent colloidal stability in biological media containing serum and were non-hemolytic to red blood cells. In vitro study confirmed that TNG could stimulate autophagy in foam cells and enhance lipid efflux and in vivo study in ApoE-/- mice indicated a significant reduction in atherosclerotic plaques, while increasing autophagic markers. In conclusion, TNG hold great promise as a trehalose delivery system to restore impaired autophagy-mediated lipid efflux in atherosclerosis and subsequently reduce atherosclerotic plaques.

Effect of cryoprotectant-induced intracellular ice formation and crystallinity on bactria during cryopreservation.

Cryopreservation is widely used for the long-term storage of bacteria. Glycerol is one of the traditional cryoprotectants used widely to prevent cryoinjury during the cryopreservation of bacteria,although it may be toxic to the cells. To overcome these issues, synthetic antifreeze polymers are also used as cryoprotectants to inhibit ice formation. In the study, we compared the performance of various antifreeze synthetic polymers including poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone), poly(ethylene glycol), and dextran with glycerol, among which PVA performed best on decreasing the ice growth rate.The impacts of glycerol, trehalose, combined with PVA on the survival of S. thermophilus were also explored. Notably,. S. thermophilus stored in 100 mg/mL trehalose and 1 mg/mL PVA +50 mg/mL trehalose combo showed significantly enhanced survival when compared with those in traditional cryoprotectant (20% [v/v] glycerol), which achieved the survival percentage of only 41.03 ± 0.09%. The effects of the freezing temperature and crystallinity on the survival of S. thermophilus were elucidated.

The response of a human haematopoietic cell line to trehalose-loaded liposomes and their effect on post-thaw membrane integrity.

Dimethyl sulphoxide (DMSO) used in haematopoietic stem cell (HSC) cryopreservation has been linked to an increased incidence of adverse reactions following transplantation. In the interest of reducing the required DMSO concentrations, we have evaluated the use of unilamellar liposomes to internalize the non-toxic, cell-impermeable disaccharide, trehalose into HSCs and characterized the cryoprotective efficacy of this strategy. A fluorescent marker, 5(6)-carboxyfluorescein (200 µmol/L), was used for trehalose internalization following a 5 h incubation at 37 °C with liposome concentrations ranging from 0.5 mM to 4 mM. Cells were frozen (1 °C/min to -80 °C) following treatment with either 3 mM or 4 mM of liposomes (5 h, 37 °C) containing 0.2 mol/L trehalose either in the presence or absence of 0.2 mol/L extracellular trehalose. Increasing the liposome concentration from 3 mM to 4 mM corresponded to a significant (p = 0.046) increase in the mean fluorescent intensity (MFI) (3 mM 512 ± 7.07; 4 mM: 916 ± 28.3). Post-thaw membrane integrity indicated that the presence of trehalose both inside and outside when internalized using a liposome concentration of 4 mM significantly improved survival relative to the sole presence of extracellular trehalose (p = 0.02). However, viability was diminished relative to a standard DMSO control (trehalose: 32.5% ± 1.7%; DMSO: 85.0% ± 4.6%). This study confirms that the protective efficacy of trehalose is enhanced when it is present on both sides of the membrane; however, it reinforces concerns surrounding the efficiency of using liposomes as a vehicle to transfer trehalose into cells.

Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro.

OBJECTIVES: Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. METHODS: HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett's and Tukey's tests (p < 0.05). RESULTS: Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. CONCLUSION: Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder. CLINICAL RELEVANCE: With the limitations of an in vitro design, our study suggests that in terms of cell response, trehalose-based air-polishing powders show a reduced effect on inflammation.

Cryopreservation of HEP-G2 cells attached to substrates: the benefit of sucrose and trehalose in combination with dimethyl sulfoxide.

BACKGROUND: Cryopreservation of mammalian cells is mainly done in cryovials as free cell suspension in 5 to10% (v/v) dimethyl sulfoxide (DMSO). Relatively little attention has been paid to cryopreservation of adherent cell monolayers. OBJECTIVE: To investigate the appropriate cryoprotectant (CPA) formulations for the cryopreservation on HEP-G2 human tumor cells attached to the polystyrene plate and plastic surfaces. MATERIALS AND METHODS: Five CPA formulations were evaluated for the cryopreservation of HEP-G2 cells attached to polystyrene plates and and plastic coverslips, using post-thaw cell viability as a performance indicator. RESULTS AND CONCLUSION: Hep-G2 cells attached to the plastic coverslips and polystyrene plate surfaces were successfully cryopreserved in 10% DMSO with sucrose and trehalose. The addition of saccharides enabled the reduction of DMSO concentration, replaced serum, and improved the functional capacity of post-thaw Hep-G2 cells. Cells attached to the plastic coverslips show significantly better results than those attached to the polystyrene plate surfaces after cryopreservation. doi.org/10.54680/fr22310110412.

Trehalose-based neuroprotective autophagy inducers.

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and small molecule prodrugs (2-5), putative probes (6-10) and inorganic nanovectors (12a - thiol-PEG-triazole-trehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing properties. While small molecules 2-11 showed no pro-autophagic behavior at sub-millimolar concentrations, trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 µM trehalose concentration without any significant toxicity at the same concentration.

Poly 2-methacryloyloxyethyl Phosphorylcholine Protects Corneal Cells and Contact Lenses from Desiccation Damage.

SIGNIFICANCE: Contact lens (CL) wearing may cause discomfort and eye dryness. We describe here the efficacy of a synthetic polymer in protecting both the corneal epithelial cells and the CL from desiccation damage. Artificial tears containing this polymer might be helpful to treat or prevent ocular surface damage in CL wearers. PURPOSE: We aimed to investigate the protective effects of the synthetic polymer 2-methacryloyloxyethyl phosphorylcholine (poly-MPC) on corneal epithelial cells and CLs subjected to desiccation damage. METHODS: The interaction of poly-MPC with the cell membrane was evaluated on human primary corneal epithelial cells (HCE-F) by the sodium dodecyl sulfate damage protection assay or the displacement of the cell-binding lectin concanavalin A (ConA). Survival in vitro of HCE-F cells and ex vivo of porcine corneas exposed to desiccating conditions after pre-treatment with poly-MPC or hyaluronic acid (HA), hypromellose (HPMC), and trehalose was evaluated by a colorimetric assay. Soft CLs were soaked overnight in a solution of poly-MPC/HPMC and then let dry in ambient air. Contact lens weight, morphology, and transparency were periodically registered until complete dryness. RESULTS: Polymer 2-methacryloyloxyethyl phosphorylcholine and HPMC were retained on the HCE-F cell membrane more than trehalose or HA. Polymer 2-methacryloyloxyethyl phosphorylcholine, HA, and HPMC either alone or in association protected corneal cells from desiccation significantly better than did trehalose alone or in association with HA. Contact lens permeation by poly-MPC/HPMC preserved better their shape and transparency than did saline. CONCLUSIONS: Polymer 2-methacryloyloxyethyl phosphorylcholine coats and protects corneal epithelial cells and CLs from desiccation damage more efficiently compared with trehalose and as good as other reference compounds.

Sulfated vizantin causes detachment of biofilms composed mainly of the genus Streptococcus without affecting bacterial growth and viability.

BACKGROUND: Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva. RESULTS: The biofilm, composed mainly of the genus Streptococcus and containing 50 µM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 µM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 µM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence. CONCLUSION: Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.

Trehalose-Conjugated, Catechin-Loaded Polylactide Nanoparticles for Improved Neuroprotection against Intracellular Polyglutamine Aggregates.

Intracellular/extracellular protein aggregation is linked to a variety of neurodegenerative diseases. Current research focuses on identifying antiamyloidogenic small molecules to inhibit such protein aggregation and associated cytotoxicity. We have recently demonstrated that transforming these antiamyloidogenic small molecules into nanoparticle forms can greatly improve their performance, and biocompatible/biodegradable formulation of such nanoparticles is critical for therapeutic applications. Here, we report polylactide (PL)-based biodegradable nanoparticles for improved neuroprotection against polyglutamine (polyQ) aggregation that is responsible for Huntington's disease. PL is terminated with an antiamyloidogenic trehalose molecule or the neurotransmitter dopamine, and the resultant nanoparticle is loaded with the antiamyloidogenic catechin molecule. The self-assembled nanoparticle is ∼200 nm in size and enters into the neuronal cell, inhibits polyQ aggregation, lowers oxidative stress, and enhances cell proliferation against polyQ aggregates. This biodegradable polymer can be used in nanoformulation of other reported antiamyloidogenic molecules for testing various animal models of neurodegenerative diseases.

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans.

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.

Simple Syntheses of New Pegylated Trehalose Derivatives as a Chemical Tool for Potential Evaluation of Cryoprotectant Effects on Cell Membrane.

In our work, we developed the synthesis of new polyfunctional pegylated trehalose derivatives and evaluated their cryoprotective effect using flow cytometry. We showed that new compounds (modified trehaloses) bound to appropriate extracellular polymeric cryoprotectants could be helpful as a chemical tool for the evaluation of their potential toxic cell membrane influences. Our aim was to form a chemical tool for the evaluation of cryoprotectant cell membrane influences, which are still not easily predicted during the freezing/thawing process. We combined two basic cryoprotectants: polyethyleneglycols (PEGs) and trehalose in the new chemical compounds-pegylated trehalose hybrids. If PEG and trehalose are chemically bound and trehalose is adsorbed on the cell surface PEGs molecules which are, due to the chemical bonding with trehalose, close to the cell surface, can remove the cell surface hydration layer which destabilizes the cell membrane. This was confirmed by the comparison of new material, PEG, trehalose, and their mixture cryoprotective capabilities.

Effects of subgingival air-polishing with trehalose powder on oral biofilm during periodontal maintenance therapy: a randomized-controlled pilot study.

BACKGROUND: This pilot study was part of a larger study which compared the effect of subgingival air-polishing using trehalose powder with sonic scaling on clinical parameters during supportive periodontal therapy. Within this microbiological part of the investigation subgingival samples were taken from 10 participants to analyze the survival of different bacterial species after the two different treatments as a proof of principle. METHODS: In 10 participants two non-adjacent, single-root teeth requiring treatment (PD =5 mm with bleeding on probing (BOP) or > 5 mm) were selected following a split-mouth design and were treated either with a sonic scaler or air-polishing device and trehalose powder. For persistent pockets (PD =4 mm and BOP or > 4 mm), treatment was repeated after 3 months. Subgingival biofilm samples were taken at baseline (BL), subsequently and three and six months after treatment. After determination of the bacterial counts (TBL), isolated bacteria were identified by MALDI-TOF-MS. If unsuccessful, PCR and 16S rDNA sequencing were performed. RESULTS: In both treatment groups, TBL decreased immediately after treatment remaining at a lower level. This confirms the findings of the larger study regarding clinical parameters showing a comparable effect on PD, BOP and CAL. Immediately after treatment, the diversity of detected species decreased significantly more than in the sonic group (p = 0.03). After 3 months, the proportion of Gram-positive anaerobic rods was lower in the air-polishing group (powder/ sonic 7%/ 25.9%, p = 0.025). Also, there was a greater reduction of Gram-negative aerobic rods for this group at this time (air-polishing/ sonic - 0.91 / -0.23 Log10 cfu/ ml, p = 0.020). CONCLUSION: Within the limitations of this study air-polishing and sonic treatment seem to have a comparable effect on the subgingival oral biofilm during supportive periodontal treatment. TRIAL REGISTRATION: The study was registered in an international trial register (German Clinical Trial Register number DRKS 00006296) on 10th of June 2015. HTML&TRIAL_ID = DRKS00006296.

Beneficial Effects of Trehalose on Striatal Dopaminergic Deficits in Rodent and Primate Models of Synucleinopathy in Parkinson's Disease.

Disease modification in Parkinson's disease (PD) is an unmet medical need. In the current study, we evaluated trehalose, a safe and well-tolerated disaccharide that has previously demonstrated efficacy in rodent models of neurodegenerative diseases, including PD. In a rat model of PD, based on delivery of adeno-associated virus serotype 1/2 containing the mutated human A53T α-synuclein gene (AAV1/2-hourA53T-aSyn) to the substantia nigra (SN), we showed that rats administered trehalose (2.67 g/kg per day, by mouth) for 6 weeks had less forelimb asymmetry (93% reduction) and higher striatal dopamine (54% increase) compared with rats receiving vehicle. In a pharmacokinetic study, we determined that efficacy was associated with plasma C max of 8900 ng/ml and area under the curve from time 0 to infinity (AUC0-inf) of 11,136 hour⋅ng/ml. We then showed, in macaques, that oral administration of trehalose (2.67 g/kg per day) produced plasma exposures of similar magnitude, with plasma C max of 10,918 ng/ml and AUC0-inf of 27,445 hour⋅ng/ml. In a macaque model of PD, also based on delivery of AAV1/2-hourA53T-aSyn to the SN, trehalose (2.67 g/kg per day, by mouth), administered for 142 days, produced higher striatal dopamine (by 39%) and dopamine transporter levels (by 50%), compared with macaques receiving vehicle. In neither model did trehalose treatment prevent loss of tyrosine hydroxylase (TH) positive (TH+ve) cells in the SN or alter α-synuclein levels in the striatum. These studies demonstrated that trehalose reduces striatal dopaminergic deficits in a rodent and macaque model of synucleinopathy in PD. Furthermore, we have determined the pharmacokinetic parameters associated with efficacy, and thus defined exposures to target in future clinical trials.

Endogenous arabitol and mannitol improve shelf life of encapsulated Metarhizium brunneum.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 ± 0.2 and 2.1 ± 0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 ± 4.4 to 55.0 ± 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 ± 3.3% at 5 °C followed by 54.5 ± 1.6% at 18 °C and 19.4 ± 1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 ± 7.6 to 98.0 ± 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Cryopreservation of Luciola praeusta Kiesenwetter (Coleoptera: Lampyridae) embryos by vitrification.

There is an urgent need to preserve the ever-decreasing number of different species of fireflies all over the world. We sought to develop a vitrification procedure to cryopreserve the firefly embryos. The late stages of Luciola praeusta Kiesenwetter embryos were collected. Several impermeable and permeable protectants with various concentrations in different mediums (TNM-FH insect medium, Grace's medium, Dulbecco's Modification of Eagle's Medium (DMEM) and Dulbecco's Phosphate-Buffered Saline (DPBS)) were used. Embryos culturing in TNM-FH medium yielded the highest survival rate of 75.3 ± 3.6%. One-step, two-step and three-step methods were used in equilibrium procedure respectively. The highest survival rate (73.7% ±3.2%) occurred in embryos treated by three-step method ((1.5 M ethylene glycol (EG) + 2 M EG plus 8% polyvinylpyrrolidone (PVP) + 3 M EG, 8% PVP and 15% trehalose). Additionally, embryos exposed to 0.5 M trehalose presented a significantly higher survival rate (71.8 ± 2.7%) than embryos preserved in 0.5 M sucrose.

The Serum-Resistant Transfection Evaluation and Long-Term Stability of Gene Delivery Dry Powder Based on Mesoporous Silica Nanoparticles and Polyethyleneimine by Freezing-Drying.

Mesoporous silica nanoparticles (MSNs) with large surface area, tunable pore size, and low toxicity can act as suitable vehicles for drug and gene delivery. An MSN/DNA/PEI complex delivery system was prepared by using MSNs to hold plasmid DNA coated with polyethyleneimine (PEI), and the dry powder formulation was produced by freeze-drying with trehalose as lyoprotectant. The MSN/DNA/PEI complexes successfully enhanced the gene expression with about 1.5-fold higher efficiency as compared with the control, and even better effects and lower toxicity were achieved at lower content of PEI. Also, this gene delivery system showed nearly sixfold higher efficiency in the serum-containing condition than the control, so further application of these vehicles in vivo is highly appreciated. Besides, the trehalose containing lyophilized formulation could hold the availability for at least 4 months of storing at room temperature, presenting the potential for industrial production and transportation of gene therapy.

Cryopreservation of lipid bilayers by LEA proteins from Artemia franciscana and trehalose.

The capacity of Late Embryogenesis Abundant (LEA) proteins and trehalose to protect liposomes against freezing-induced damage was examined by measuring the leakage of 5(6)-carboxyfluorescein (CF). Liposomes were prepared to simulate the lipid compositions of the inner leaflet of the plasma membrane, outer mitochondrial membrane (OMM), and inner mitochondrial membrane (IMM). Two recombinant LEA proteins belonging to Group 3 (AfrLEA2 and AfrLEA3m) were expressed and purified from embryos of Artemia franciscana. Only OMM-like liposomes were significantly protected by AfrLEA2 and AfrLEA3m against freeze-thaw damage; at the highest protein:lipid mass ratio tested, leakage of CF was 56.3% of control with AfrLEA3m and 29.3% with AfrLEA2. By comparison, trehalose provided protection to all compositional types. The greatest stabilization during freezing occurred when trehalose was present on both sides of the bilayer. When mitochondria isolated from rat liver were freeze-thawed in trehalose solution, the OMM remained intact based on the absence of increased oxygen consumption when cytochrome c was added during oxidative phosphorylation (OXPHOS). Respiratory control ratios (OXPHOS/LEAK) were depressed by only 30% after freeze-thawing in trehalose compared to non-frozen controls, which indicated some retention of OXPHOS capacity by the IMM. Trehalose then was loaded into the matrix (0.24 µmol/mg mitochondrial protein) by transient opening of the permeability transition pore, a procedure optimized for retention of OMM integrity. Surprisingly, respiratory control ratios were not improved after freeze-thawing with external plus matrix trehalose, when compared to external trehalose alone. This result could perhaps be explained by insufficient accumulation of matrix trehalose.

Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.