Absence Of Cryoprotectants Research Articles

Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines.

The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at -20°C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.

Cryoprotectant-free preservation of bacteria using semi-spherical drops

Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities. However, freeze-thaw damage and osmotic pressure changes during cryopreservation adversely impacts microbial survival. Significant expenditure of resources and expertise are required to select the right cryoprotectant and optimize its concentration for maximum survival of diverse microorganisms. This work describes a cryopreservation method that obviates the need for cryoprotectants by exploiting the unique thermal characteristics of semi-spherical drops. Here, a plurality of these drops, each 10 μl in volume, created on a highly non-wetting flat-sheet substrate with holes and frozen at −70 °C. Deriving an f (x) metric as a measure of relative viability, storage in drops in the absence of cryoprotectants was found to improve the survivability of Staphylococcus epidermidis by 1.91 times compared with the same sample stored in larger 50-μl volumes in standard 1.5-ml tubes. This also compares well with a value of 2.33 obtained with standard preservation with cryoprotectant. The drop method allows high throughput aliquoting of the bacterial culture into multiple discrete drops using multichannel pipettes or automated liquid handlers and the edges of the holes provides a pinning action that holds the drop stably against gravitational roll-offs. It also allows samples to be removed in discrete small volumes, thus, reducing the number of freeze thaw cycles and associated cell damage. The flat-sheet architecture of the substrate reduces the amount of plastic waste generated and augments green laboratory practices.

Freeze-drying of monoclonal antibody-conjugated gold nanorods: Colloidal stability and biological activity

Maintaining colloidal stability of nanoparticles in suspensions is a major challenge. Therefore, freeze-drying (lyophilization) is recently proposed to preserve colloidal stability of nanoparticles through maintaining them in a solid state. However, freeze-drying would itself induce nanoparticle aggregation unless proper formulation with a careful selection of cryoprotectants is considered. Herein, we evaluate the colloidal stability of gold nanorods (GNRs) conjugated with a rituximab as a model monoclonal antibody upon freeze-drying in the presence of various cryoprotectants (mannitol, trehalose and sucrose). Aggregation-induced optical responses of GNRs were used as a sensitive tool to follow nanoparticle aggregation. In the absence of cryoprotectants, rituximab-conjugated GNRs aggregate irreversibly while evaluated cryoprotectants exhibit a significant protective effect. Maximal colloidal stability of GNRs is observed in the presence of trehalose while mannitol results in best cake formation in terms of shape and integrity. A combination of trehalose and mannitol produces a lyophilized product with satisfactory GNR colloidal stability and cake shape. Moreover, we show that freeze-dried rituximab-conjugated GNRs in presence of proper cryoprotectants maintain typical binding to lymphoma tissues as confirmed via immunohistochemistry assay.

HDL structure and function is profoundly affected when stored frozen in the absence of cryoprotectants

Analysis of structural and functional parameters of HDL has gained significant momentum in recent years because they are stronger predictors of cardiovascular risk than HDL-cholesterol levels. Surprisingly, in most HDL studies, very low attention is paid to HDL storage, which might critically affect functional properties. In the present study, we systematically examined the impact of storage and freezing on the structural/functional properties of freshly isolated HDL. Initial damage to HDL starts between week 1 and week 4 of storage. We observed that prolonged freezing at −20°C or −70°C led to a shedding of apoA-I from HDL and to the formation of large protein-poor particles, indicating that HDL is irreversibly disrupted. These structural alterations profoundly affected key metrics of HDL function, including HDL-cholesterol efflux capacity and HDL paraoxonase activity. Flash-freezing of isolated HDL prior to storage at −70°C did not preserve HDL structure. However, addition of the cryoprotectants, sucrose or glycerol, completely preserved structure and function of HDL when stored for at least 2 years. Our data clearly indicate that HDL is a complex particle requiring special attention when stored. Addition of cryoprotectants to isolated HDL samples before storage will make biochemical and clinical HDL research studies more reproducible and comparable.

Porcine sperm vitrification I: cryoloops method.

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n=3; r=4) and raw (n=5; r=2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20×106 spermatozoa ml-1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30s at 37°C) and an ultra-rapid method (7s at 75°C, followed by 30s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

Impact of alginate concentration on the viability, cryostorage, and angiogenic activity of encapsulated fibroblasts

Cryopreservation or cryostorage of tissue engineered constructs can enhance the off-the shelf availability of these products and thus can potentially facilitate the commercialization or clinical translation of tissue engineered products. Encapsulation of cells within hydrogel matrices, in particular alginate, is widely used for fabrication of tissue engineered constructs. While previous studies have explored the cryopreservation response of cells encapsulated within alginate matrices, systematic investigation of the impact of alginate concentration on the metabolic activity and functionality of cryopreserved cells is lacking. The objective of the present work is to determine the metabolic and angiogenic activity of cryopreserved human dermal fibroblasts encapsulated within 1.0%, 1.5% and 2.0% (w/v) alginate matrices. In addition, the goal is to compare the efficacy of dimethyl sulfoxide (DMSO) and trehalose as cryoprotectant. Our study revealed that the concentration of alginate plays a significant role in the cryopreservation response of encapsulated cells. The lowest metabolic activity of the cryopreserved cells was observed in 1% alginate microspheres. When higher concentration of alginate was utilized for cell encapsulation, the metabolic and angiogenic activity of the cells frozen in the absence of cryoprotectants was comparable to that observed in the presence of DMSO or trehalose.

14 EPIGENETIC CHANGES IN EQUINE SPERMATOZOA DURING CRYOPRESERVATION: A PRELIMINARY STUDY

The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure. However, semen processing may also contribute to epigenetic changes in spermatozoa. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. For this purpose, semen was collected from fertile Shetland pony stallions. Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100 ng of DNA per sample). The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Statistical analysis was done with the SPSS Statistics 21 software. Values are means ± standard error of the mean. In Experiment 1, 1.5 mL of raw semen (n = 6 stallions, 1 ejaculate each) was shock-frozen at –196°C for 15 min and thawed at 38°C for 60 s. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In Experiment 2, 3 ejaculates per stallion (n = 6) were included. Semen quality and DNA-methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (Minitube, Tiefenbach, Germany; cryoprotectant: 5% glycerol) or BotuCrio (Nidacon, Mölndal, Sweden; cryoprotectants: 1% glycerol and 4% methylformamid) extender. Semen was frozen in 0.5-mL straws in a computer-controlled rate freezer (IceCube 14 M; Sylab, Purkersdorf, Austria, cooling rates: 20°C to 5°C: 0.3°C min–1, 5°C to 25°C: 10°C min–1, –25°C to –140°C: 25°C min–1). Semen motility, morphology, and membrane integrity were significantly reduced (e.g. total motility before freezing: 88.8 ± 1.4%) by cryopreservation but not influenced by the extender used (e.g. total motility: Ghent 69.5 ± 2.0, BotuCrio 68.4 ± 2.2%; P < 0.001 v. nonfrozen semen). Cryopreservation significantly (P < 0.01) increased the level of DNA-methylation (before freezing: 0.6 ± 0.1%, Ghent 6.4 ± 3.7, BotuCrio 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA-methylation was not correlated with semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA-methylation level in stallion semen. In the present study, this effect occurred irrespective of the cryoprotectant but was not seen after shock-freezing in the absence of cryoprotectants. The reduced fertility of mares after insemination with frozen-thawed semen may at least in part be explained by methylation of sperm DNA, which occurs in response to the cryopreservation procedure.

Effect of supercooling and cell volume on intracellular ice formation

Intracellular ice formation (IIF) has been linked to death of cells cryopreserved in suspension. It has been assumed that cells can be supercooled by 2 to 10°C before IIF occurs, but measurements of the degree of supercooling that cells can tolerate are often confounded by changing extracellular temperature and solutions of different osmolality (which affect the cell volume). The purpose of this study was to examine how the incidence of IIF in the absence of cryoprotectants is affected by the degree of supercooling and cell volume. Human umbilical vein endothelial cells were suspended in isotonic (300mOsm) and hypertonic (∼600 to 700mOsm) solutions and exposed to supercooling ranging from 2 to 10°C before extracellular ice was nucleated. The number of cells undergoing IIF was examined in a cryostage (based on the darkening of cells upon intracellular freezing (“flashing”)) as a function of the degree of supercooling, and cell survival post-thaw was assessed using a membrane integrity assay. We found that while the incidence of IIF increased with supercooling in both isotonic and hypertonic solutions, it was higher in the isotonic solution at any given degree of supercooling. Since cells in hypertonic solution were shrunken due to water efflux, we hypothesized that the difference in IIF behavior could be attributed to the decreased volume of cells in the hypertonic solution. Our results confirm that cells with a smaller diameter before extracellular ice nucleation have a decreased probability of IIF and suggest that cell volume could play a more significant role in the incidence of IIF than the extracellular ice nucleation temperature.

Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop.

Cryotop is a carrier that has been used successfully in the cryopreservation of human spermatozoa. Here, we explored a novel method to vitrify human spermatozoa without cryoprotective agents (CPAs) using Cryotop. Spermatozoa from 21 Normozoospermic patients were collected and vitrified without CPAs or with sucrose in small volume using Cryotop. The sperm recovery rate, motility, viability, chromatin damage and DNA fragmentation were assessed. No significant difference was observed in the sperm recovery rate and motility rate between the spermatozoa cryopreserved without CPAs and with sucrose. The post-thawed spermatozoa cryopreserved without CPAs had a higher viability and lower damage to sperm chromatin and DNA than those cryopreserved with sucrose. These results suggest that small numbers of human spermatozoa can be successfully vitrified without CPAs using Cryotop.

Effects of Deep Hypothermic Preservation on Posttransplant Viability of Tracheal Grafts

Objective Using an animal model, we performed a study to assess the effects of rapid freezing in the absence of cryoprotectants on the viability of tracheal grafts after allotransplantation. Materials and methods Ten tracheal heterotopic transplantations were performed with fresh grafts just after harvesting donors, and 20, with cryopreserved specimens at 2 or 6 weeks after hypothermic storage. Cryopreservation consisted of rapid cooling in the absence of cryoadditives with storage for 2 weeks at −80°C. After rewarming, grafts were transplanted into the abdominal cavity of immunosuppressed rats by being embedded into the greater omentum. Fourteen days later, the implants were removed to examine chondrocyte viability, submucosal swelling, and epithelial patterns in comparison with fresh controls. Results All allografts retained their macroscopic structure after transplantation. Respiratory epithelium was substituted by planar epithelium only in the frozen transplanted specimens. Fresh transplanted grafts exhibited 100% chondral viability in contrast with 5% viability observed among cryopreserved tracheas after transplantation. Conclusions The method of rapid freezing was effective to preserve airway grafts. However, the quality of cryopreserved cartilage was disappointing, similar to that obtained using conventional slow-freezing protocols.

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen–thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20–30% yielded the most motile sperm. Cooling at a slow rate (29°C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen–thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220°C/min, and thawed rapidly in a 37°C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.

Freeze drying of human serum albumin (HSA) nanoparticles with different excipients

Freeze drying is a suitable technique to improve the long-term storage stability of colloidal drug carrier systems such as nanoparticles. Aim of this study was to systematically evaluate excipients for the freeze drying and long-term stability of albumin-based nanoparticles. In our study, nanoparticles made of human serum albumin (HSA) were freeze dried in the presence of different cryoprotective agents and after reconstitution were evaluated with regard to their physico-chemical characteristics. Empty, doxorubicin-loaded, and PEGylated nanoparticles were prepared and were freeze dried in the presence of different concentrations of sucrose, trehalose, and mannitol, respectively. The samples were physico-chemically characterised with regard to lyophilisate appearance, particle size, and polydispersity using photon correlation spectroscopy. For evaluation of long-term stability, the samples were stored at 2–8, 25, and 40 °C over predetermined time intervals. In the absence of cryoprotectants, particle growth was observed in all freeze-dried formulations. In the presence of sucrose, mannitol, and trehalose aggregation of HSA nanoparticles during the freeze-drying procedure was prevented. Although all of the excipients were identified to be suitable stabilisers for freeze drying of HSA nanoparticles, sucrose and trehalose were superior to mannitol, especially with regard to the long-term storage stability results.

Long term storage of virus templated fluorescent materials for sensing applications

Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studiedfor long term preservation in the presence and absence of cryoprotectants. Viral complexeswere reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNaseassay for structural integrity. When viruses lyophilized in the absence of cryoprotectantwere rehydrated and RNase treated, UV absorption increased, indicating that the capsidswere damaged. The addition of trehalose during lyophilization protected capsid integrityfor at least 7 weeks. Measurements of the fluorescence peak maximum of CPMV lyophilizedwith trehalose and reconstituted also indicate that the virus remained intact. Microarraybinding assays indicated that CPMV particles chemically modified for use as a fluorescenttracer were intact and retained binding specificity after lyophilization in the presence oftrehalose. Thus, we demonstrate that functionalized CPMV nanostructures can bestored for the long term, enabling their use in practical sensing applications.

A New Device for Mechanical Testing of Blood Vessels at Cryogenic Temperatures

As part of an ongoing program to study the thermo-mechanical effects associated with cryopreservation via vitrification (vitreous in Latin means glassy), the current study focuses on the development of a new device for mechanical testing of blood vessels at cryogenic temperatures. This device is demonstrated on a bovine carotid artery model, permeated with the cryoprotectant cocktail VS55 and a reference solution of 7.05M DMSO, below glass transition. Results are also presented for crystallized specimens, in the absence of cryoprotectants. Results indicate that the elastic modulus of a specimen with no cryoprotectant, at about -140°C (8.6°C and 15.5°C below the glass transition temperature of 7.05M DMSO and VS55, respectively), is 1038.8 ± 25.2 MPa, which is 8% and 3% higher than that of a vitrified specimen permeated with 7.05M DMSO and VS55, respectively. The elastic modulus of a crystallized material at -50°C is lower by ~20% lower from that at -140°C.

Thermal expansion of blood vessels in low cryogenic temperatures Part I: A new experimental device

As part of the ongoing effort to study the mechanical behavior of biological material during cryopreservation processes, the current study focuses on thermal expansion of blood vessels at low cryogenic temperatures. The current paper (Part I) describes a new experimental device for thermal expansion measurements of blood vessels in typical conditions of vitrification, which are associated with rapid cooling rates. For validation purposes, the thermal strain of frozen arteries in the absence of cryoprotectants was measured, and found to be about 10% larger than that of polycrystalline water; this observation agrees with literature data. The companion paper (Part II) reports on experimental results of cryoprotectants permeated with VS55, DP6, and 7.05 M DMSO at high cooling rates applicable to vitrification.

Computational Fluid Dynamics Analysis of Cell Cooling Process

Vitrification is the solidification of a solution without crystallization, caused by an increase of viscosity. It results from a balance between the highest realizable cooling rates and the highest concentrations of cryoprotectants (CPA) not toxic for the cells for the period of exposure. To vitrify with reduced CPA concentrations the cooling rates have to be increased. The objective of our study was to verify the thermal feasibility of a vitrification process using a computational fluid dynamics (CFD) model. The first step in our stepwise approach to our project is focused on an ultrarapid freezing process in absence of cryoprotectants. The main parameters which influence the cooldown and increased cooling rate inside the biologic material (e.g., the thermophysical parameters, the size and the shape of the specimen) have been investigated. In our test cases the living material is considered frozen by direct contact with a surface at cryogenic temperature. Both numerical values and false color images of the thermal fields are available. It is confirmed that higher cooling rates can be obtained by decreasing the thickness of the material placed on the cold surface or changing the cryogen. Variations of the other thermophysical parameters had little influence on the freezing time. From the results it appears that it is impossible to define only one cooling rate for specimen vitrification and that large quantities of biological material could be ultrarapidly frozen on wide cold surfaces with low material thickness.

Modified vitrification and cooling of human pronuclear oocytes: efficacy and effect on ultrastructure

The efficacy of cryopreservation by direct plunging into liquid nitrogen (vitrification) of human pronuclear oocytes using open pulled straws with a super-finely pulled tip, as well as the ultrastructural changes caused by cooling and vitrification, were evaluated. Clinical and electron microscopic studies of cooled and vitrified oocytes were performed. Oocytes were cooled to 4°C in the presence and absence of cryoprotectants, vitrified, warmed, cultured and transferred. Abnormally fertilized oocytes were examined by electron microscopy. Vitrified and warmed 2-pronuclear oocytes showed 71.1% survival rate and 83.3% developmental rate. One- and 3-pronuclear oocytes, after cooling without cryoprotectants (presumably non-viable), showed progressive swelling of mitochondrial smooth endoplasmic reticulum (SER). After vitrification, oocytes (presumably viable) showed the formation of large SER vesicles associated with mitochondria. The described protocol of vitrification of human pronuclear oocytes was shown to be effective in producing pregnancy. Normal ultrastructure after undergoing the described vitrification protocol was confirmed.

Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success

The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

Development of a new cyclosporine formulation based on poly(caprolactone) microspheres

The present study describes the development of a new cyclosporine formulation based on polycaprolactone (PCL) microspheres (MS) prepared by the solvent evaporation method. Ternary phase diagrams were used to identify the domains where MS were formed. The application of central composite designs established the influence of several technological (stirring speed) and formulation factors (polymer and surfactant amounts, and organic solvent volume) on the size of PCL MS. Cyclosporine-loaded MS of a size around 2.5 #181;m were prepared and characterized. The stability of the systems, either alone or loaded with cyclosporine, stored at 8#176;C and room temperature (RT) was assessed as well. Freeze-drying was evaluated as an alternative method to achieve longterm stability. The experimental design showed that the stirring speed and the organic phase volume were the only parameters significantly affecting the MS size. Experimental conditions selected to obtain CyA-loaded MS of 2.5 #181;m resulted in a high entrapment percentage (98.4 - 0.66%), with the drug dissolved or molecularly dispersed within the dense polymeric matrix of MS. After 12 months of storage at 8#176;C and RT, PCL MS remained physically stable, although the crystallinity of the polymer increased by 35% upon storage at both temperatures. Freeze-drying studies revealed that MS could be successfully lyophilized in the absence of cryoprotectants without significant changes of the drug entrapment; however, the presence of at least 5% cryoprotectant was essential to keep the initial particle size. Therefore, a stable MS-based CyA formulation was easily prepared and characterized. This formulation offer the possibility of CyA administration through different routes.

Structural changes in natural actomyosin and surimi from ling cod (Ophiodon elongatus) during frozen storage in the absence or presence of cryoprotectants.

Surimi and natural actomyosin (NAM) from ling cod (Ophiodon elongatus) were subjected to frozen storage in the absence or presence of cryoprotectants (sorbitol, sucrose, lactitol, and Litesse, either individually or in combination). Effects of frozen storage were studied for NAM frozen at -10 degrees C for 10 days and for surimi after eight freeze-thaw cycles. A commercial blend cryoprotectant (4% sucrose and 4% sorbitol), individual cryoprotectants at 8%, and optimal blends at 4, 5.5, 6, and 8%, were effective in maintaining the gel strength of surimi and NAM gels. Surimi or NAM frozen in the absence of cryoprotectants or with only 4% individual cryoprotectants, showed increased percent alpha-helical content by Raman analysis. Increased disulfide content was also observed in the treatment without cryoprotectants by the Raman SS stretching band and by chemical determination. Tyrosine residues were in a buried environment before and after freezing for all treatments, and surface hydrophobicity measured by 1-anilinonaphthalene-8-sulfonate decreased after frozen storage in the absence of cryoprotectants.