Cryoprotective Effect Research Articles

Dynamic intervention to enhance the stability of PEGylated Ibrutinib loaded lipidic nano-vesicular systems: transitioning from colloidal dispersion to lyophilized product.

Liposomes being a promising colloidal system facilitates delivery of drugs with limited pharmacokinetic properties to achieve desirable clinical applications. However, development of a stable liposomal system is always challenging due to multiple complexities involved. Aqueous instability of liposomes and impact of various process and formulation parameters can lead to serious alteration of its therapeutic performance. In the proposed work, the authors aim to develop stable Ibrutinib-loaded liposomes using lyophilization and Quality-by-Design and assess their long-term stability. Ibrutinib-loaded liposomes were developed and optimized using Quality-by-Design technique and were further PEGylated and characterized for the same. Effect of cryoprotectants during lyophilization and other parameters are evaluated to obtain a robust formulation. The stability studies were conducted upto 6 months at various storage conditions to evaluate the effect of lyophilization. The impact of formulation, processing and lyophilization parameters on physicochemical properties of developed liposomal systems were evaluated and are critically discussed. Liquid dispersion exhibited a %degradation of 16-36% at 25°C/60% RH which was reduced for less than 1% in lyophilized formulation for 6 months. Critical analysis and assessment of various parameters lead to identification of optimum conditions to manufacture this drug product and also opens way forward for further evaluation and translational possibilities.

Boron Facilitates Amelioration of Hepatic Injury by the Osmolyte Glycine and Resolves Injury by Improving the Tissue Redox Homeostasis

Background: Glycine is a conditional non-essential amino acid in human and other mammals. It is abundant in the liver and is known for a wide spectrum of characteristics including the antioxidant, antiinflammatory, immunomodulatory, and cryoprotective effects. The amino acid is a naturally occurring osmolyte compatible with protein surface interactions and has been reported in literature as a potent therapeutic immuno-nutrient for liver diseases such as alcoholic liver disease. Oral glycine administration protects ethanol-induced liver injury, improves serum and tissue lipid profile, and alleviates hepatic injury in various conditions. In recent years, sodium salt of boron (borax) has been reported for its beneficial effects on cellular stress, including the effects on cell survival, immunity, and tissue redox state. Incidentally both glycine and boron prevent apoptosis and promote cell survival under stress. Objective: This study investigates the beneficial effect of borax on liver protection by glycine. Methods: Briefly, liver toxicity was induced in rats by a single intraperitoneal injection of thioacetamide (400 mg/kg b. wt.). Results: Significant changes in oxidative stress and liver function test parameters, the molybdenum Fe-S flavin hydroxylase activity, nitric oxide and tissue histopathology were observed in thioacetamide treated positive control group. The changes were ameliorated both by glycine as well as borax, but the combinatorial treatment yielded a better response indicating the impact of boron supplementation on glycine mediated protection of liver injury in experimental animal model. Conclusions: The study has clinical implications as the hepatotoxicity caused by thioacetamide mimics features of hepatitis C infection in human.

Evaluation of Kappa-carrageenan supplementation in extender for post-thaw Kajli ram sperm quality.

Cryopreservation is one of the reliable techniques for long-term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa-carrageenan (κ-carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non-colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k-carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k-carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post-thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p < .05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k-carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k-carrageenan improved the sperm post-thaw quality.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

An effective strategy for improving the freeze-drying survival rate of Lactobacillus curvatus and its potential protective mechanism

Freeze-drying is a common method to prepare dried starter cultures with high stability and a long storage time. However, the bacterial cells suffer varying degrees of damage during the freeze-drying process. This study aimed to provide an effective strategy to improve the survival rate of Lactobacillus curvatus and to explore its potential protective mechanisms. The survival rates of bacterial cells in different cryoprotectants before and after freeze-drying were measured using the plate count method. The protective effects of composite cryoprotectants on bacterial cells were investigated from multiple perspectives, including key enzyme activities, the internal morphology of bacterial cells, and cell membrane integrity. Results showed that the freeze-drying survival rate of bacterial cells in the composite cryoprotectants consisting of trehalose, oleic acid and skimmed milk (85.38%) was significantly improved compared to that in PBS solution (2.04%). The addition of composite cryoprotectants could reduce damage to the cell wall and cell membrane, maintain the normal morphology of bacterial cells and cell membrane integrity, and improve key enzyme activities (lactate dehydrogenase, pyruvate kinase and ATPases) during the freeze-drying process. The viability of freeze-dried cultures was also monitored at different temperatures during storage, and the cell survival of freeze-dried cultures at −20 °C was the highest, reaching 61.13%. The findings obtained in this work offer a new method to improve the viability of Lactobacillus curvatus after freeze-drying, and may promote the wide use of freeze-dried cultures in the food fermentation industry.

Inhibition of protein denaturation and oxidation of prepared shrimp paste by sturgeon skin collagen peptide

This study investigated the cryoprotective effect of different concentrations (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%) of sturgeon skin collagen peptides (CPs) on the changes in protein denaturation, oxidation, and overall quality of prepared shrimp paste during 180 d cryopreservation. Compared with phosphates and sorbitol, higher concentrations (>1.5%) of CPs minimized the denaturation and oxidation of protein with higher protein solubility, thermal transition enthalpy, and less carbonyl formation. Meanwhile, the decrease of hydrogen and ionic bonding was inhibited accompanied by less thawing loss and cooking loss. Compared with sorbitol, CPs at all concentrations contributed to less antioxidant effect in retarding the lipid oxidation after 180 d, however still exhibited lower thiobarbituric acid values than the control. The freshness of the 2.5% CPs group and sorbitol group was well maintained with the total volatile basic nitrogen value kept under 20 mg/100 g. Furthermore, a compact and less-aggregated gel matrix was observed during frozen storage. These findings indicate that CPs posed dose-dependent cryoprotective effects on shrimp paste, representing it as a comparable alternative to polyphosphates or sorbitol for seafood cryopreservation.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Effect of Five Different Antioxidants on the Effectiveness of Goat Semen Cryopreservation.

The effective combination of semen cryopreservation and artificial insemination has a positive effect on the conservation of germplasm resources, production and breeding, etc. However, during the process of semen cryopreservation, the sperm cells are very susceptible to different degrees of physical, chemical, and oxidative stress damage. Oxidative damage is the most important factor that reduces semen quality, which is affected by factors such as dilution equilibrium, change of osmotic pressure, cold shock, and enzyme action during the freezing-thawing process, which results in the aggregation of a large amount of reactive oxygen species (ROS) in sperm cells and affects the quality of semen after thawing. Therefore, the method of adding antioxidants to semen cryoprotective diluent is usually used to improve the effect of semen cryopreservation. The aim of this experiment was to investigate the effects of adding five antioxidants (GLP, Mito Q, NAC, SLS, and SDS) to semen cryoprotection diluent on the cryopreservation effect of semen from Saanen dairy goats. The optimal preservation concentrations were screened by detecting sperm viability, plasma membrane integrity, antioxidant capacity, and acrosomal enzyme activities after thawing, and the experimental results were as follows: the optimal concentrations of GLP, Mito Q, NAC, SLS, and SDS added to semen cryopreservation diluent at different concentrations were 0.8mg/mL, 150nmol/L, 0.6mg/mL, 0.15mg/ mL, 0.6mg/mL, and 0.15mg/mL. The optimal concentrations of the five antioxidants were added to the diluent and analyzed after 1week of cryopreservation, and it was found that sperm viability, plasma membrane integrity, and mitochondrial activity were significantly enhanced after thawing compared with the control group (P < 0.05), and their antioxidant capacity was significantly enhanced (P < 0.05). Therefore, the addition of the above five antioxidants to goat sperm cryodilution solution had a better enhancement of sperm cryopreservation. This study provides a useful reference for exploring the improvement of goat semen cryoprotection effect.

Plant Cryopreservation: Principles, Applications, and Challenges of Banking Plant Diversity at Ultralow Temperatures.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

Technology for obtaining fermented products based on walnut milk

A technology has been developed for obtaining lyophilized fermented products based on walnut milk. Fermentation was carried out with two probiotic strains of Lactiplantibacillus plantarum subsp. plantarum NBIMCC 3447 and Lactobacillus gasseri NBIMCC 2450. In the capacity of prebiotic and cryoprotectant the composition of the products includes fructooligosaccharides (FOS) in three concentrations - 1, 2 and 4%. An increase in cell survival was found with an increase in the concentration of the fructo-oligosaccharides in the medium. After fermentation and lyophilization, L. plantarum showed better survival compared to L. gasseri in all samples examined. Probiotic cells fermented in medium with 4% FOS retain high viability after lyophilization - 93-96%, while those in medium with 2% FOS reach 85-93% survival. Fructooligosaccharides not absorbed by the cells at the end of the fermentation process render a cryoprotective effect and lead to a higher survival of the cells of both strains in the lyophilization process.

Cryoprotective effect of curdlan on frozen wheat gluten: With respect to physicochemical properties and molecular structure

Curdlan alleviates the quality deterioration of frozen dough during storage by a mechanism related to retarded migration of moisture. However, the strengthening effect of curdlan on the gluten structure needs to be proved. Therefore, the effect of curdlan on physicochemical properties and molecular structure changes of frozen wheat gluten was investigated. Frozen storage resulted in decreased water-holding capacity and viscoelasticity properties, as well as increase in oil-holding capacity and surface hydrophobicity. However, curdlan significantly inhibited the rheological and physicochemical properties changes of gluten. Scanning electron microscope results showed that the structure of gluten with curdlan had relatively smoother and less damaged surface during frozen storage. Curdlan alleviated the depolymerization of gluten macropolymers (GMP) by restraining the breakage of S–S bonds, with GMP depolymerization degree of 20% after 90 days of storage, significantly lower than that of the control (38%). Curdlan restrained the transition of α-helix to β-turn and antiparallel β-sheet, and suppressed the protein unfolding and chromophores exposure. Curdlan interacted with gluten prominently by hydrogen bonds, followed by hydrophobic interactions, thus strengthening the gluten structure. Alleviation of dough quality loss during frozen storage by curdlan could be related to its strengthening gluten structure.

The cryoprotective effect of Litopenaeus vannamei head-derived peptides and its ice-binding mechanism

Although discarded as waste, shrimp heads are a potential source of antifreeze peptides, which can be used as cryoprotectants in the food industry. Their utilization in frozen foods can help mitigate the negative effects caused by the freezing technique. Litopenaeus vannamei shrimp heads were autolyzed, and the shrimp head autolysate (SHA) was separated via ultra-filtration and ion exchange chromatography. The antifreeze effect of SHA on the biochemical properties of myofibrillar proteins of peeled shrimps during five freeze-thaw cycles was evaluated. Peptide screening was done using the LC-MS/MS technique. A molecular docking (MD) study of the interaction between ice and shrimp head-derived antifreeze peptides was done. Results showed that shrimp-head autolysate has a maximum thermal hysteresis value of 1.84 °C. During the freeze-thaw cycles, the shrimp-head autolysate exhibited an antifreeze effect on frozen peeled shrimps. 1.0 and 3.0%-SHA groups showed significantly lower freeze denaturation than the negative control group. The muscle tissues of SHA-treated groups were not as severely damaged as the negative control group. The molecular docking study revealed that the shrimp head-AFPs bound to ice via hydrogen bonding, and both hydrophilic and hydrophobic amino acid residues were involved in the ice-binding interactions. 6 ice-binding sites were involved in the peptide-ice interaction. Our findings suggest that shrimp head-derived AFPs can be developed into functional additives in frozen foods and add more insights into the existing literature on antifreeze peptides.

Molecular modelling studies reveal cryoprotective mechanism of L-Proline during the frozen storage of shrimp (Litopenaeus vannamei)

This study aimed to investigate the cryoprotective properties of proline (1% and 3% (w/v)) on shrimp. The cryoprotective mechanism was studied using physico-chemical experiments and molecular simulations. Proline had a notable positive impact on the thawing loss and texture of shrimp in comparison to the control. The denaturation of myosin in frozen shrimp was delayed by proline. Microscopy analysis demonstrated that proline effectively lowered the harm caused by ice crystals to shrimp muscle. Molecular simulations indicated that proline potentially exerted a cryoprotective effect primarily through the “water substitution” and “glassy state” hypotheses. Proline formed hydrogen bonds with myosin to replace the water molecules around myosin. Additionally, proline interacted with water molecules to form a glassy state, impeding the growth of ice crystals. Consequently, the stability of shrimp myosin was enhanced during freezing. In conclusion, proline demonstrated promise as an efficacious cryoprotectant for aquatic products.

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation.

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

Cryostorage of peripheral blood hematopoietic stem cells in transplantology: current status and prospects

Peripheral blood hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of hematological, cancer and autoimmune diseases. In cancer patients, HSC transplantation allows the use of high-dose cytotoxic drugs in combination with radiation therapy during treatment, which provides a pronounced antitumor effect. The hematological toxicity of such treatment is eliminated by the sequential introduction of stem cells, which contribute to hematopoiesis restoration. Before transplantation, peripheral blood HSCs are subjected to collection and cryopreservation for further storage. An important requirement for cryopreservation is viable HSCs responsible for hematopoietic restoration. The aim of the review was to analyze the literature devoted to the influence of various methods of cryopreservation of human peripheral blood HSCs on the preservation of cell viability after thawing, as well as the development of adverse events in patients. Issues related to the use of various cryoprotectants, as well as methods for storing HSC grafts, are considered. The presented data indicate the need for further study of the effect of cryoprotectants on the human body and the cellular composition of the graft and improvement of protocols for HSC cryopreservation.

48 Effect of different cryoprotectants and slow freezing on viability of Saiga (Saiga tatarica) fibroblasts

48 Effect of different cryoprotectants and slow freezing on viability of Saiga (Saiga tatarica) fibroblasts

Effects of preservative solution and cryoprotectant on living rats

Effects of preservative solution and cryoprotectant on living rats

Transitioning from a research protocol to a scalable applied pathway for Xenopus laevis sperm cryopreservation at a national stock center: The effect of cryoprotectants.

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center-level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 106 sperm/mL) on post-thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10%DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%-13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species.

Cryoprotective effect of soybean oil on surimi gels and the mechanism based on molecular dynamics simulation.

The effect of soybean oil (SO) on freeze-thaw (F-T)-treated surimi was investigated and its related mechanism was revealed by molecular dynamics (MD) simulations. The results displayed that SO has a disrupting effect on the structure of fresh samples. However, in the F-T-treated samples, surimi gels supplemented with SO had a more uniform microstructure. Simultaneously, when SO was added from 0% to 7% in the F-T-treated samples, the gel strength increased from (p < .05), the physically bound water was increased from 92.90% to 94.15% (p < .05), and storage modulus was increased from 5939 to 6523 Pa. Triglycerides of SO generated hydrophobic interactions with myosin mainly in carbon chains. Computational results from MD simulations illustrated that the structure of myosin combined with triglycerides was more stable than that of myosin alone during temperature fluctuations (-20 to 4°C). During ice crystal growth, triglycerides absorbed on the myosin surface inhibited the growth of surrounding ice crystals and mitigated the ice crystal growth rate (from 7.54 to 5.99 cm/s). The addition of SO during the F-T treatments allowed myosin to be less negatively affected by ice crystal formation and temperature fluctuations and ultimately contributed to the formation of a more uniform network gel structure.

Effect of Cryoprotectants on Quality of Desalted Jellyfish Subjected to Multiple Freeze- Thaw Cycles

A freeze-thaw cycle in frozen products occurs when the temperature fluctuates during storage or transportation, causing drip loss, changes in ice crystal reformation, and textural protein. In practical freezing, using cryoprotectants in frozen products aids in delaying the physicochemical changes. The problem has been found in commercial frozen jellyfish with sesame oil, causing the separating oil and water derived from drip loss of thawed jellyfish protein. This study aimed to select an appropriate cryoprotectant and concentration for frozen jellyfish products. Therefore, this research compared the changes in the physical and textural properties of desalted jellyfish collagen protein soaked in inulin, sucrose, or sorbitol at 1, 5, and 10% and subjected to three freeze-thaw cycles. Results showed increased concentration of each cryoprotectant increased soaking yield. The maximum soaking yields of desalted jellyfish were 2.49 ± 0.54, 2.79 ± 0.82, and 2.78 ± 0.51%, and each cryoprotectant content was 7.18 ± 0.01, 7.54 ± 0.00, and 8.58 ± 0.32% when using static soaked in inulin, sucrose, and sorbitol at 10%. During the freeze-thaw cycle, the retardation of the denatured jellyfish protein from ice crystals increased when desalted jellyfish were immersed in inulin, sucrose, or sorbitol at the maximum concentration of 10%, displaying the drip losses at 27.88 ± 0.45, 29.45 ± 0.35, and 28.56 ± 0.73% that lowered than the control at 56.54 ± 0.64%. The increased repeated freeze-thaw cycles increased the compact structure of thawed jellyfish collagen, supported by microstructure analysis. In summary, inulin at 10% appears to have a cryoprotective effect similar to sucrose and sorbitol and will be a choice for commercial frozen jellyfish-based food menu development.