Effective Cryoprotective Agent Research Articles

Insights into Chemical Interactions and Related Toxicities of Deep Eutectic Solvents with Mammalian Cells Observed Using Synchrotron Macro–ATR–FTIR Microspectroscopy

Deep eutectic solvents (DESs) and ionic liquids (ILs) are highly tailorable solvents that have shown a lot of promise for a variety of applications including cryopreservation, drug delivery, and protein stabilisation. However, to date, there is very limited information on the detailed interactions of these solvents with mammalian cells. In this work, we studied six DESs and one IL that show promise as cryoprotective agents, applying synchrotron macro–ATR–FTIR to examine their effects on key biochemical components of HaCat mammalian cells. These data were paired with resazurin metabolic assays and neutron reflectivity experiments to correlate cellular interactions with cellular toxicity. Stark differences were observed even between solvents that shared similar components. In particular, it was found that solvents that are effective cryoprotective agents consistently showed interactions with cellular membranes, while high toxicity correlated with strong interactions of the DES/IL with nucleic acids and proteins. This work sheds new light on the interactions between novel solvents and cells that may underpin future biomedical applications.

Hyaluronic acid as effective cryoprotective agent for hMSC cryopreservation

Hyaluronic acid as effective cryoprotective agent for hMSC cryopreservation

Deep eutectic solvents as cryoprotective agents for mammalian cells.

Cryopreservation has facilitated numerous breakthroughs including assisted reproductive technology, stem cell therapies, and species preservation. Successful cryopreservation requires the addition of cryoprotective agents to protect against freezing damage and dehydration. For decades, cryopreservation has largely relied on the same two primary agents: dimethylsulfoxide and glycerol. However, both of these are toxic which limits their use for cells destined for clinical applications. Furthermore, these two agents are ineffective for hundreds of cell types, and organ and tissue preservation has not been achieved. The research presented here shows that deep eutectic solvents can be used as cryoprotectants. Six deep eutectic solvents were explored for their cryoprotective capacity towards mammalian cells. The solvents were tested for their thermal properties, including glass transitions, toxicity, and permeability into mammalian cells. A deep eutectic solvent made from proline and glycerol was an effective cryoprotective agent for all four cell types tested, even with extended incubation prior to freezing. This deep eutectic solvent was more effective and less toxic than its individual components, highlighting the importance of multi-component systems. Cells were characterised post-thawing using atomic force microscopy and confocal microscopy. Molecular dynamics simulations support the biophysical parameters obtained by experimentation. This is one of the first times that this class of solvents has been systematically tested for cryopreservation of mammalian cells and as such this research opens the way for the development of potentially thousands of new cryoprotective agents that can be tailored to specific cell types. The demonstrated capacity of cells to be incubated with the deep eutectic solvent at 37 °C for hours prior to freezing without significant loss of viability is a major step toward the storage of organs and tissues.

Cryopreservation of mammalian cells using protic ionic liquid solutions

Cryopreservation has facilitated considerable advances in both medical technology and scientific research. However, further developments have been limited by the relatively low number of effective cryoprotective agents. Even after fifty years of research, most protocols rely on the same two toxic agents, i.e. dimethylsulfoxide or glycerol. Ionic liquids are a class of promising solvents which are known glass formers and may offer a less-toxic alternative. The research presented here investigates ten protic ionic liquids as potential cryoprotective agents. The liquids are screened for key properties including cellular toxicity, permeability and thermal behaviour. The most promising, ethylammonium acetate, was then tested as a cryoprotective agent on a model cell line and was found to be as effective as the common cryoprotectant, dimethylsulfoxide. This work reports the first use of a protic ionic liquid as an effective cryoprotective agent for a mammalian cell line. This will inform the development of a suite of potential new ionic liquid-based cryoprotectants that could potentially allow the cryopreservation of new cell types.

Cryopreservation and Transplantation of Laboratory Rodent Ovarian Tissue for Genome Banking and Biomedical Research.

Genetic modifications in combination with highly sophisticated assisted reproductive technologies such as in vitro oocyte maturation and development, in vitro fertilization, intracytoplasmic sperm injection, and in vitro embryo culture have opened many research avenues and treatment options for both animals and humans. The number of genetically modified (GM) rodent strains increased considerably during the last several decades, and their numbers are expected to increase due to efficient gene editing technologies including the CRISPR/Cas9. Rodent ovarian tissues (OT) cryopreservation and transplantation procedures have several applications in biomedical field: they provide a fertility restoration option for GM rodent strains in some circumstances. They also serve as models to investigate OT cryopreservation as potential alternatives for human infertility patients as well as other domestic and wildlife species for the development of improved cryopreservation and subsequent transplantation strategies. The modeling studies enable determining effective cryoprotective agents (CPA), CPA and water permeability kinetics, and cooling and warming rates during the development of OT cryopreservation procedures. Furthermore, rodent models are extremely useful for determining post-thaw OT graft sites as well as potential medical interventions in an effort to expedite angiogenesis and inhibit inflammatory/immune response, OT longevity, and follicular integrity. Here we describe methodologies for rodent OT cryopreservation and potential transplantation sites for frozen-thawed rat and mouse OT.

The Historic Development and Incorporation of Four Assisted Reproductive Technologies Shaping Today′s IVF Industry

The concept of assisted reproductive technologies was most notably derived from the late 19th Century experiments of Sir Walter Heape who successfully transplanted rabbit embryos. Interestingly, it was not until the late 1940’s and 1950’s that renewed interests in rabbit embryo transfer and cryobiology occurred. The history behind developing effective procedures can be fascinating, though few could be more accidental than Dr. Chris Polge’s discovery of glycerol (1948), from a mislabeled bottle of sugar solutions, being an effective cryoprotective agent for sperm freezing. The purpose of this review paper is to discuss four key scientific breakthrough technologies occurring between 1985 and 1995, which ultimately shaped the future of today’s human in vitro fertilization (IVF) industry. More importantly, this paper highlights the foundation of underlying related discoveries and some unique stories involving their development and publication. In the end, this paper emphasizes the value of understanding scientific discovery timetables and the eventual re-discovery in the hands and minds of creative, determined and dedicated scientists, as history tends to repeat itself before its useful application is realized.

The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography.

Both crystallization and cryoprotection are often bottlenecks for high-resolution X-ray structure determination of macromolecules. Methylamine osmolytes are known stabilizers of protein structure. One such osmolyte, trimethylamine N-oxide (TMAO), has seen occasional use as an additive to improve macromolecular crystal quality and has recently been shown to be an effective cryoprotective agent for low-temperature data collection. Here, TMAO and the related osmolytes sarcosine and betaine are investigated as primary precipitating agents for protein crystal growth. Crystallization experiments were undertaken with 14 proteins. Using TMAO, seven proteins crystallized in a total of 13 crystal forms, including a new tetragonal crystal form of trypsin. The crystals diffracted well, and eight of the 13 crystal forms could be effectively cryocooled as grown with TMAO as an in situ cryoprotective agent. Sarcosine and betaine produced crystals of four and two of the 14 proteins, respectively. In addition to TMAO, sarcosine and betaine were effective post-crystallization cryoprotective agents for two different crystal forms of thermolysin. Precipitation reactions of TMAO with several transition-metal ions (Fe(3+), Co(2+), Cu(2+) and Zn(2+)) did not occur with sarcosine or betaine and were inhibited for TMAO at lower pH. Structures of proteins from TMAO-grown crystals and from crystals soaked in TMAO, sarcosine or betaine were determined, showing osmolyte binding in five of the 12 crystals tested. When an osmolyte was shown to bind, it did so near the protein surface, interacting with water molecules, side chains and backbone atoms, often at crystal contacts.

Assessment of strategies to increase chondrocyte viability in cryopreserved human osteochondral allografts: evaluation of the glycosylated hydroquinone, arbutin

Allogeneic cartilage is used to repair damaged areas of articular cartilage, requiring the presence of living chondrocytes. So far, no preservation method can effectively meet that purpose. Identification of more effective cryoprotective agents (CPAs) can contribute to this goal. The aim of this study was to determine whether the glycosylated hydroquinone, arbutin, alone or in combination with low concentrations of other CPAs, has cryoprotective properties towards human articular cartilage. Human tibial plateaus were procured from multi-organ donors, with the approval of the Ethics Committee of the University Hospital of Coimbra. The tibial plateaus were treated with or without arbutin (50 or 100mM), alone or in combination with various concentrations of dimethyl sulfoxide (DMSO) and glycerol, for 0.5-1.5h/37 degrees C, then frozen at -20 degrees C and 24h later transferred to a biofreezer at -80 degrees C. Two to 3 months later, thawing was achieved by immersion in cell culture medium at 37 degrees C/1h. Chondrocyte viability was assessed before and after freeze-thawing using a colorimetric assay based on the cell's metabolic activity and fluorescent dyes to evaluate cell membrane integrity. Before freezing, chondrocyte metabolic activity was identical in all the conditions tested. After freeze-thawing, the highest activity, corresponding to 34.2+/-2.1% of that in the Fresh Control, was achieved in tibial plateaus incubated in 50mM arbutin for 1h whereas in those left untreated it was 11.1+/-4.7. Addition of DMSO and glycerol to arbutin did not increase chondrocyte viability any further. Fluorescence microscopy confirmed these results and showed that living chondrocytes were mainly restricted to the superficial cartilage layers. Arbutin seems to be an effective cryoprotective agent for osteochondral allografts with potential benefits over DMSO and glycerol.

Interaction between advancing ice fronts and erythrocytes

It can be shown theoretically and experimentally that in purely aqueous suspension, cells (as well as microsolutes) are excluded by advancing freezing fronts. This puts the cells under considerable osmotic stress and may be considered to be the major source of cell destruction upon freezing. It is also shown theoretically and experimentally that in aqueous suspensions, admixed with appropriate concentrations of a cryoprotectant (e.g., glycerol), cells are engulfed by advancing freezing fronts: Under such conditions, cells do not undergo any osmotic stress and remain undamaged when frozen. The influence of various common cryoprotectants is discussed, as is the reason why penetrating as well as nonpenetrating agents can be equally effective cryoprotective agents. The reason why leukocytes require lower cryoprotectant concentrations than erythrocytes is also elucidated.

Quick freezing of mouse embryos using ethylene glycol with lactose or sucrose

Mouse compacted morulae were quickly frozen in liquid nitrogen (LN 2) vapor at approximately −170°C after 5 min equilibration in 2 M or 3 M ethylene glycol or glycerol in the presence of either 0.25 M lactose or sucrose to examine their post-thaw viability. After thawing in a water bath at 37°C and a one-step dilution of the cryoprotectant, the embryos were cultured for 48 h. Significantly higher in vitro survival rates were obtained with 3 M ethylene glycol than with 2 M ethylene glycol or 2 or 3 M glycerol irrespective of the sugar used ( P < 0.001). However, 2 M ethylene glycol with lactose gave a significantly higher survival rate than in combination with sucrose ( P < 0.05). On the contrary, 3 M glycerol with sucrose gave a significantly higher survival rate than when combined with lactose ( P < 0.001). The transfer of frozen-thawed embryos cryopreserved with 3 M ethylene glycol with 0.25 M lactose or 0.25 M sucrose resulted in the development of normal fetuses at rates of 51.7% and 47.7%, respectively, as compared to 73.8% for fresh embryos. This study suggests that a higher concentration of ethylene glycol in combination with lactose or sucrose is an effective cryoprotective agent during quick freezing.

Rheological Properties of Fresh and Frozen Chum Salmon Eggs With and Without Treatment by Cryoprotectants

ABSTRACTChum salmon eggs from six batches of ovaries were subjected to flat plate compression loading to determine the membrane strength. The intact eggs withstood compression forces of between 0.47 to 1.22 newtons. The membrane rupture energy ranged from 2.36 to 7.99 (x 10‐4) joules. After freezing fresh eggs at ‐10°C for 7 days, the average membrane rupture energy upon thawing was 46% less than that for fresh eggs. The apparent viscosities of fresh and frozen‐thawed yolk of chum eggs were similar, and it may be concluded that yolk proteins were not cryodamaged. Sodium chloride was found to be the most effective cryoprotective agent for the prevention of membrane cryodamage.

Postfreeze viability of human renal epithelial carcinoma cells in quaternary solutions

The postfreeze survival of human renal epithelial carcinoma cells frozen in suspensions based on (MEM + FCS) (Eagle's Minimum Essential Medium with Hanks' salts added and supplemented with 20 vol% heat-inactivated fetal calf serum) to which additions of NaCl and the cryoprotective compounds, dimethyl sulfoxide and glycerol, were made, have been determined by a vital dye exclusion technique. A significant range of aqueous-rich quaternary solutions were found to be highly effective cryoprotective agents for these cells under optimum freezing and thawing conditions. High survival occurs in a composition series emanating from the region of high postfreeze cell viability in the ternary (MEM + FCS)-NaCl-DMSO system, between 80 and 95% (MEM + FCS), along isopleths that substitute glycerol for NaCl in various proportions. In addition, viability remains sufficiently high as a few percentage of glycerol is substituted for DMSO alone.

Preservation of Avian Cells at Sub-zero Temperatures

The cryoprotective agents dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone (PVP) and dextran were evaluated for their ability to protect avian cells during storage at sub-zero temperatures. DMSO was the most effective cryoprotective agent for the short- and long-term storage of avian cells and glycerol was also effective when used at low concentrations. PVP and dextran did not protect avian cells during storage in our experiments. Primary chicken cells and avian cells at higher passage levels were successfully recovered after storage with DMSO for periods ranging from 4 to 12 months.

Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen‐thawed mouse eggs

Frozen mouse eggs were examined to determine the effects of low temperatures, concentration of cryoprotective agents and cooling rates on their survival, fertilizability in vitro and subsequent development. Dimethyl sulfoxide administered at 1.5 M concentration was found to be the most effective cryoprotective agent. Upon thawing, 51% and 56% of the eggs appeared to be normal after having been cooled at 0.33 degrees C/min to -30 degrees C and -50 degrees C, but only 18% of the eggs appeared to be normal after having been cooled at the same rate to -75 degrees C. When eggs were cooled at 0.33 degrees C/min to -45 degrees C and the cooling rate increased to 1 degree C/min from -45 degrees C to -75 degrees C, 44% and 72% appeared normal upon thawing. Of the normal eggs fertilized in vitro from C3H mice, 65% cleaved to the 2-cell stage and 24% of the 2-cell eggs developed into blastocysts. Following the transfer of 17 blastocysts into three recipient mice, one mouse delivered three normal young.

Effect of oxygen on freezing damage: III. Modification by β-mercaptoethylamine

The effect of β-mercaptoethylamine (MEA) on the oxygen-freezing-effect observed in E. coli strains was studied. In E. coli B s − 1, a repair deficient strain, MEA reversed the increased radiation sensitivity characteristic of the oxygen-freezing-effect. MEA also affected the number and type of free radicals in frozen bacteria, including the generation of a typical “organosulfur” radical under certain conditions. MEA caused similar free radical effects in E. coli B/r which does not show an oxygen-freezing-effect. These results support the hypothesis that the oxygen-freezing effect is mediated by free radical reactions and that E. coli B/r can repair such damage. It is suggested that the enhancement of freezing damage by oxygen could play an important role in some types of cryotoxicity and that MEA or other free radical reactants would be effective cryoprotective agents under such circumstances.

Hydroxyethyl starch: extracellular cryophylactic agent for erythrocytes.

Studies in vitro indicate that hydroxyethyl starch is an effective extracellular cryoprotective agent for erythrocytes. It is as effective as polyvinylpyrrolidone in cryophylactic ability. It is degraded to glucose units in the circulation, is not retained in tissues, and is inexpensive to produce. Changes in the size and degree of hydroxyethylation of the branched-chain starch may result in a more effective cryoprotective agent.

Studies on the survival of the rotifer philodina after freezing and thawing

Summary The survival of the rotifer Philodina after freezing and thawing has been examined under a variety of experimental conditions. Particular emphasis was placed on obtaining maximum survival by employing the most advantageous combination of protective additive concentration and cooling rate. Glycerol is an effective cryoprotective agent in this system at concentrations as low as 1% and exerts a maximum protective influence at a concentration of 2 to 3%. Glycerol solutions in the range of 5 to 10% result in somewhat anomalous survival curves as a function of cooling rate, although survival at these concentrations is moderately high at the extremes of the “slow cooling” range. Treatment of rotifers with a 20% glycerol solution results in death; thus, it is possible that sublethal toxic effects play a role at lower glycerol concentrations. Cooling rate optima appear in the region of 3 to 5°C per min with deviations occurring at high levels of glycerolation. Survivals of the order of 50 to 60% can be routinely achieved by employing the most suitable pretreatment and cooling rate. Animals frozen in this manner have been stored up to 20 days at liquid nitrogen temperatures without a noticeable decline in viability. In addition to manifesting normal swimming and feeding habits, survivors of freezing appear to enjoy a normal life span and reproductive capacity.