Cryobiology最新文献

Organs cryopreserved by vitrification are exposed to the lowest possible concentration of cryoprotectants for the least time necessary to successfully avoid ice formation. Faster cooling and warming rates enable lower concentrations and perfusion times, reducing toxicity. Since warming rates necessary to avoid ice formation during recovery from vitrification are typically faster than cooling rates necessary for vitrification, warming speed is a major determining factor for successful vitrification. Dielectric warming uses an oscillating electric field to directly heat water and cryoprotectant molecules inside organs to achieve warming that's faster and more uniform than can be achieved by heat conduction from the organ surface. This work studied 27 MHz dielectric warming of rabbit kidneys perfused with M22 vitrification solution. The 27 MHz frequency was chosen because its long wavelength and penetration depth are suitable for human organs, because it had an anticipated favorable temperature of maximum dielectric absorption in M22, and because it's an allocated frequency for industrial and amateur use with inexpensive amplifiers available. Previously vitrified kidneys were warmed from −100 °C by placement in a 27 MHz electric field formed between parallel capacitor plates in a resonant circuit. Power was varied during warming to maintain constant electric field amplitude between the plates. Maximum power absorption occurred near −70 °C, with a peak warming rate near 150 °C/min in 50 mL total volume with approximately 500 W power. After some optimization, it was possible to warm ∼13 g vitrified kidneys with unprecedentedly little injury from medullary ice formation and a favorable serum creatinine trend after transplant. Distinct behaviors of power absorption and system tuning observed as a function of temperature during warming are promising for non-invasive thermometry and future automated control of the warming process at even faster rates with user-defined temperature dependence.

This study examined the effects of liquid nitrogen vapor on osteogenesis in the rabbit femur. Cryotweezers made of porous nickel titanium alloy (nitinol or NiTi) obtained by self-propagating high temperature synthesis were used in this experiment. The porous structure of the cryotweezers allows them to hold up to 10 g of liquid nitrogen after being immersed for 2 min, which completely evaporates after 160 s. To study the effects of liquid nitrogen evaporation on osteogenesis, a rabbit femur was perforated. The formed holes were subjected to cryotherapy with varying exposure times. It was found that a 3 s exposure time stimulates osteogenesis, which was manifested in a greater number of osteoblasts in the regenerate compared to the control sample without liquid nitrogen. It was observed that increasing the exposure to 6, 9 or 12 s had a destructive effect, to varying degrees. The most severe damage was exerted by a 12 s exposure, which resulted in the formation of osteonecrosis areas. In the samples exposed to 6 and 9 s of cryotherapy, destruction of the cytoplasm of osteocytes and osteoclasts was observed.

The study is devoted to the effect of lowered resuscitation temperature (26 °C) on cryopreserved porcine adrenal glands functional activity in vitro and in vivo under xenotransplantation. The adrenals were collected from newborn pigs, cryopreserved with 5 % DMSO at a rate of 1 °C/min, resuscitated at 26 or 37 °C for 48 h (5 % CO₂, DMEM), embedded into small intestinal submucosa, and transplanted to bilaterally adrenalectomized rats. It has been shown that the glands resuscitated at 26 °C have suppressed free-radical processes and can produce cortisol and aldosterone in vitro, and may lead to elevated blood levels of these hormones. Moreover, the adrenal grafts maintain blood glucose levels and promote the formation of glycogen stores. Thus, the resuscitation at 26 °C can improve the quality of grafts and favor the introduction and application of the cryopreserved organs and tissues for transplantation in clinical and experimental practice.

Refreezing the remaining genetic resources after in vitro fertilization (IVF) can conserve genetic materials. However, the precise damage inflicted by repeated freezing and thawing on bovine sperm and its underlying mechanism remain largely unexplored. Thus, this study investigates the impact of repeated freeze-thaw cycles on sperm. Our findings indicate that such cycles significantly reduce sperm viability and motility. Furthermore, the integrity of the sperm plasma membrane and acrosome is compromised during this process, exacerbating the advanced apoptosis triggered by oxidative stress. Additionally, transmission electron microscopy exposed severe damage to the plasma membranes of both the sperm head and tail. Notably, the “9 + 2” structure of the tail was disrupted, along with a significant decrease in the level of the axonemal protein DNAH10, leading to reduced sperm motility. IVF outcomes revealed that repeated freeze-thaw cycles considerably impair sperm fertilization capability, ultimately reducing the blastocyst rate. In summary, our research demonstrates that repeated freeze-thaw cycles lead to a decline in sperm viability and motility, attributed to oxidative stress-induced apoptosis and DNAH10-related dynamic deficiency. As a result, the utility of semen is compromised after repeated freezing.

Nowadays, the physical nature of supersaturated binary aqueous sugar solutions in the vicinity of the glass transition represents a very important issue due to their biological applications in cryopreservation of cells and tissues, food science and stabilization and storage of nano genetic drugs. We present the construction of the Supplemented Phase Diagram and the non-equilibrium nature of the undersaturated-supersaturated kinetic transition. The description of its thermodynamic nature is achieved through the study of behavior of their viscosity as temperature is lowered and concentration increased. In this work, we find a universal character for the viscosities of several sugar water solutions.

In the 1970s, sperm cryopreservation was presented as a unique route to fertility preservation. The ability to cryopreserve sperm from all species is challenging. The sperm cryopreservation process encompasses various cellular stresses such as increased osmotic pressure, ice crystal formation, and thermal shock, therefore decreasing the quality of sperm. The nanostructures due to their inherent features such as reactivity, high uptake, active surface area, and antioxidant activity, have contributed to modifying freezing protocols. In this review, the current state of the art with regards to emerging applications of nanotechnology in sperm cryopreservation are reviewed, some of the most promising advances are summarized, and the limitations and advantages are comprehensively discussed.

Nowadays the significant role of biobanks in medical, diagnostic, industrial, and environmental research is well known. Bacterial biobanks could be used as a good resource for designing new treatments, biomedical and industrial researches, and laboratory diagnostics. To have a collection of bacteria from clinical samples and maintain their long-term viability, their preservation needs appropriate protective agents, like cryoprotectants and lyoprotectants. In this study, we collected and characterized Gram-negative and Gram-positive bacteria carrying important antibiotic resistance markers from different clinical samples of hospitalized children. Sucrose (10%), skimmed milk (10%), skimmed milk plus sodium glutamate (10% + 1%), and bovine serum albumin (BSA, 10%) were used as lyoprotectants during the freeze-drying procedure. The survival rate of the lyophilized samples was calculated by dilution plating and measuring the colony forming unit (CFU) after 3 months of storage. The culture analysis results indicated that 25 of the 27 studied bacterial genera (Dilutions 10⁻³ to 10⁻⁶), including Shigella, Methicillin-resistant S. aureus, Acinetobacter spp., Escherichia spp., Pseudomonas spp., Klebsiella spp., Enterococcus spp., were recovered in cultured fractions from all preservation conditions, while 2 genera were only detected in a single preservation condition (2/27, 7.4%). Based on the results, sucrose (10%) and skimmed milk (10%) presented the most protective features. The survival rates varied significantly according to types of the bacteria. Collectively, our results showed a diversity in the recovery of different bacterial genera after lyophilization. While statistically no significant difference was detected among the studied protective agents, sucrose (10%) and skimmed milk (10%) exhibited more effective lyoprotective properties for both Gram-positive and Gram-negative bacteria among the clinical isolates in our study.

Human induced pluripotent stem (hiPS) cells have demonstrated promising potential in regenerative medical therapeutics. After successful clinical trials, the demand for hiPS cells has steadily increased. Therefore, the optimization of hiPS cell freezing processes for storage and transportation is essential. Here, we presented a computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for hiPS cells. This study was based on a model that calculates cell survival rates after thawing, and the model was extended to evaluate cell potentials until 24 h after seeding. To estimate parameter values for this extension, freezing experiments were performed using constant cooling rates. Using quality and productivity indicators, we evaluated 16,206 temperature profiles using our model, and a promising profile was obtained. Finally, an experimental investigation of the profile was undertaken, and the contribution of the temperature profile to both quality and productivity was confirmed.

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 10⁶ spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

Chimeric antigen receptor (CAR) T-cell therapies are increasingly adopted as a commercially available treatment for hematologic and solid tumor cancers. As CAR-T therapies reach more patients globally, the cryopreservation and banking of patients’ leukapheresis materials is becoming imperative to accommodate intra/inter-national shipping logistical delays and provide greater manufacturing flexibility. This study aims to determine the optimal temperature range for transferring cryopreserved leukapheresis materials from two distinct types of controlled rate freezing systems, Liquid Nitrogen (LN2)-based and LN2-free Conduction Cooling-based, to the ultracold LN2 storage freezer (≤−135 °C), and its impact on CAR T-cell production and functionality. Presented findings demonstrate that there is no significant influence on CAR T-cell expansion, differentiation, or downstream in-vitro function when employing a transfer temperature range spanning from −30 °C to −80 °C for the LN2-based controlled rate freezers as well as for conduction cooling controlled rate freezers. Notably, CAR T-cells generated from cryopreserved leukapheresis materials using the conduction cooling controlled rate freezer exhibited suboptimal performance in certain donors at transfer temperatures lower than −60 °C, possibly due to the reduced cooling rate of lower than 1 °C/min and extended dwelling time needed to reach the final temperatures within these systems. This cohort of data suggests that there is a low risk to transfer cryopreserved leukapheresis materials at higher temperatures (between −30 °C and −60 °C) with good functional recovery using either controlled cooling system, and the cryopreserved materials are suitable to use as the starting material for autologous CAR T-cell therapies.