The Effect of Choline Salt Addition to Trehalose Solution for Long-Term Storage of Dried and Viable Nuclei from Fully Grown Oocytes.

Although drying techniques are exciting alternatives to cryopreservation, it remains challenging to maintain tightly controlled temperatures and humidity levels during storage of dried products. The objective of this study was to determine if the addition of choline acetate to trehalose solution could enable a wider range of storage conditions for preservation of nuclei from fully grown oocytes, by allowing temporary humidity excursions (>44% relative humidity) that may lead to crystallization of trehalose and loss of DNA integrity. Using domestic cat germinal vesicle oocytes as a model, we characterized the recovery as well as the integrity of samples after microwave-assisted dehydration. Exposure to choline acetate alone did not impair the germinal vesicle's DNA integrity and only had a negative impact on the chromatin configuration. Choline acetate addition enabled us to reach lower moisture contents after 25 min of microwave-assisted drying. Sample recovery after rehydration was also better in the presence of choline acetate. The integrity of the germinal vesicle's DNA was not affected, while the chromatin configuration was impaired by the presence of choline acetate during dehydration. Importantly, choline acetate addition helped to maintain an amorphous state (absence of detrimental crystallization) during excursion from ideal humidity conditions.

Preserving the Female Genome in Trehalose Glass at Supra-Zero Temperatures: The Relationship Between Moisture Content and DNA Damage in Feline Germinal Vesicles.

INTRODUCTION: Maintaining a stable dry state is critical for long-term preservation of live biomaterials at suprazero temperatures. The objective of the study was to characterize the effect of moisture content on DNA integrity within the germinal vesicle (GV) of feline oocytes following dehydration and storage at 22-24 °C. METHODS: Using microwave-assisted drying, conditions that led to a predictable and stable moisture content in trehalose solutions were determined. To explore moisture content stability during storage, trehalose samples were dried for 15 min and stored in glass vials at 11 or 43% RH for 8 weeks. To examine whether this condition allowed proper storage of GVs, permeabilized cat oocytes were incubated in trehalose for 10 min and dried for 15 or 30 min. Oocytes then were rehydrated to assess DNA integrity either directly after drying or after 8 weeks of storage in an 11% RH environment. Raman spectroscopy was used to identify the states of dried samples during storage. RESULTS: Moisture content was stable during the storage period. There was no significant difference in DNA integrity between fresh and dried samples without storage. After 8 weeks of storage, DNA integrity was maintained in GVs dried for 30 min. Samples dried for 15 min and stored were compromised, suggesting crystallization of the preservation matrix during storage. Biostabilization was optimal when samples were directly processed to moisture contents consistent with storage in the glassy state. CONCLUSION: Microwave-assisted drying processing and storage conditions were optimized to ensure stable long-term storage of structural and functional properties of genetic resources.

Microwave- and Laser-Assisted Drying for the Anhydrous Preservation of Biologics.

Dry preservation has become an attractive approach for the long-term storage of biologics. By removing water from the matrix to solidify the sample, refrigeration needs are reduced, and thus storage costs are minimized and shipping logistics greatly simplified. This chapter describes two energy deposition technologies, namely, microwave and laser systems, that have recently been used to enhance the rate and nature of solution densification for the purpose of anhydrous preservation of feline oocytes, sperm, and egg white lysozyme in trehalose glass. Several physical screening methodologies used to determine the suitability of an amorphous matrix for biopreservation are also introduced in this chapter.

Light-assisted drying for anhydrous preservation of biological samples: optical characterization of the trehalose preservation matrix.

Protein-based drugs have been developed to treat a variety of conditions and assays use immobilized capture proteins for disease detection. Freeze-drying is currently the standard for the preservation of proteins, but this method is expensive and requires lengthy processing times. Anhydrous preservation in a trehalose amorphous solid matrix offers a promising alternative to freeze-drying. Light assisted drying (LAD) is a processing method to create an amorphous trehalose matrix. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation accelerates drying and as water is removed the trehalose forms a protective matrix. In this work, LAD samples are characterized to determine the crystallization kinetics of the trehalose after LAD processing and the distribution of amorphous trehalose in the samples. These characteristics influence the long-term stability of the samples. Polarized light imaging revealed that LAD processed samples are stable against crystallization during low-humidity storage at room temperature. Scanning white light interferometry and Raman spectroscopy indicated that trehalose was present across samples in an amorphous form. In addition, differential scanning microcalorimetry was used to measure the thermodynamic characteristics of the protein lysozyme after LAD processing. These results demonstrate that LAD does not change the properties of this protein.

Structure and Function of Porcine Arteries Are Preserved for up to 6 Days Using the HypoRP Cold-storage Solution.

BACKGROUND: Maintaining functional vessels during preservation of vascularized composite allografts (VCAs) remains a major challenge. The University of Wisconsin (UW) solution has demonstrated significant short-term benefits (4-6 h). Here we determined whether the new hypothermic resuscitation and preservation solution HypoRP improves both structure, survival, and function of pig arteries during storage for up to 6 days. METHODS: Using porcine swine mesenteric arteries, the effects of up to 6-day incubation in a saline (PBS), UW, or HypoRP solution on the structure, cell viability, metabolism, and function were determined. RESULTS: After incubation at 4°C, for up to 6 days, the structures of the arteries were significantly disrupted, especially the tunica media, following incubation in PBS, in contrast with incubation in the HypoRP solution and to a lesser extent, in UW solution. Those disruptions were associated with increased active caspase 3 indicative of apoptosis. Additionally, while incubation in PBS led to a significant decrease in the metabolic activity, UW and HypoRP solutions allowed a stable to increased metabolic activity following 6 days of cold storage. Functional responsiveness to phenylephrine (PE) and sodium nitroprusside (SNP) decreased over time for artery rings stored in PBS and UW solution but not for those stored in HypoRP solution. Moreover, artery rings cold-stored in HypoRP solution were more sensitive to ATP. CONCLUSIONS: The HypoRP solution improved long-term cold storage of porcine arteries by limiting structural alterations, including the collagen matrix, reducing apoptosis, and maintaining artery contraction-relaxation functions for up to 6 days.

Synergistic Development of Biochips and Cell Preservation Methodologies: A Tale of Converging Technologies.

PURPOSE OF THE REVIEW: Over the past several decades, cryopreservation has been widely used to preserve cells during long term storage, but advances in stem cell therapies, regenerative medicine, and miniaturized cell-based diagnostics and sensors are providing new targets of opportunity for advancing preservation methodologies. The advent of microfluidics-based devices is an interesting case in which the technology has been used to improve preservation processing, but as the devices have evolved to also include cells, tissues, and simulated organs as part of the architecture, the biochip itself is a desirable target for preservation. In this review, we will focus on the synergistic co-development of preservation methods and biochip technologies, while identifying where the challenges and opportunities lie in developing methods to place on-chip biologics on the shelf, ready for use. RECENT FINDINGS: Emerging studies are demonstrating that the cost of some biochips have been reduced to the extent that they will have high utility in point-of-care settings, especially in low resource environments where diagnostic capabilities are limited. Ice-free low temperature vitrification and anhydrous vitrification technologies will likely emerge as the preferred strategy for long-term preservation of bio-chips. SUMMARY: The development of preservation methodologies for partially or fully assembled biochips would enable the widespread distribution of these technologies and enhance their application.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model.

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function.

Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures.

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.

Effects of Water on Structure and Dynamics of Trehalose Glasses at Low Water Contents and its Relationship to Preservation Outcomes.

Dry preservation of biologics in sugar glasses is regarded as a promising alternative to conventional cryopreservation. Evidence from various studies has suggested that there is a critical range of water content beyond which the viability of preserved biologics can be greatly compromised. In this study the viability of T-cells was determined as a function of end water content after microwave-assisted drying in trehalose solutions. Hydrogen-bonding and clustering phenomena in trehalose solutions of the same moisture content were also evaluated using molecular dynamics simulation. Post-rehydration viability decreased dramatically within the range of 0.1-1 gH2O/gdw. Molecular modeling revealed that as the water content approached 0.1 gH2O/gdw the matrix formed a large interconnected trehalose skeleton with a minimal number of bound water molecules scattered in the bulk. The diffusion coefficients of trehalose oxygen atoms most distant from the glycosidic linkage fluctuated around 7.5 × 10(-14) m(2)/s within the range of 0.02-0.1 gH2O/gdw and increased again to ~1.13 × 10(-13) m(2)/s at 0.01 gH2O/gdw and below due to the loss of water in the free volume between trehalose molecules. These insights can guide the optimal selection of final moisture contents to advance dry preservation methods.

Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further (p>0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased (p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased (p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled (p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable (p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.

Local minimum in fragility for trehalose/glycerol mixtures: implications for biopharmaceutical stabilization.

Approximately a decade ago it was observed that adding a small amount (5 wt %) of glycerol to trehalose could substantially improve the stability of enzymes stored in these glasses even though the final glass transition temperature (Tg) was reduced by ∼20 K. This finding inspired great interest in the fast dynamics of dehydrated trehalose/glycerol mixtures, leading to the observation that suppression of fast dynamics was optimal in the presence of ∼5 wt % of glycerol. It was also recognized that the fast dynamics should, in theory, be related to the fragility of these glass formers, but experimental confirmation of this hypothesis has been lacking for trehalose/glycerol mixtures or any other mixtures of this nature. In the present study a dynamic mechanical analyzer (DMA) was used to determine both the Tg and the kinetic fragility index (m) of trehalose/glycerol mixtures within the mass fraction range of 80-100 wt % of trehalose. It was found that the fragility index correlated with the mass fraction of trehalose in a nonmonotonic manner, with a local minimum between 87.5 and 95 wt % of trehalose, whereas the composition dependence of Tg was found to follow a Gordon-Taylor-like relationship, with no local minimum. The composition of 5-12.5 wt % glycerol in trehalose thus yielded a matrix that maximized the strong glass-forming contribution of glycerol, while minimizing its Tg lowering effect. This quantitative evidence supports speculation about the fragility characteristics of these mixtures that has been ongoing for the past decade. The DMA-based Tg and fragility determination method developed in this study represents a new approach for identifying optimal compositions for preservation of biologics.

Distinctly Different Glass Transition Behaviors of Trehalose Mixed with Na2HPO 4 or NaH 2PO 4: Evidence for its Molecular Origin.

PURPOSE: The present study is aimed at understanding how the interactions between sugar molecules and phosphate ions affect the glass transition temperature of their mixtures, and the implications for pharmaceutical formulations. METHODS: The glass transition temperature (Tg) and the α-relaxation temperature (Tα) of dehydrated trehalose/sodium phosphate mixtures (monobasic or dibasic) were determined by differential scanning calorimetry and dynamic mechanical analysis, respectively. Molecular dynamics simulations were also conducted to investigate the microscopic interactions between sugar molecules and phosphate ions. The hydrogen-bonding characteristics and the self-aggregation features of these mixtures were quantified and compared. RESULTS: Thermal analysis measurements demonstrated that the addition of NaH2PO4 decreased both the glass transition temperature and the α-relaxation temperature of the dehydrated trehalose/NaH2PO4 mixture compared to trehalose alone while both Tg and Tα were increased by adding Na2HPO4 to pure trehalose. The hydrogen-bonding interactions between trehalose and HPO4(2-) were found to be stronger than both the trehalose-trehalose hydrogen bonds and those formed between trehalose and H2PO4(-). The HPO4(2-) ions also aggregated into smaller clusters than H2PO4(-) ions. CONCLUSIONS: The trehalose/Na2HPO4 mixture yielded a higher T g than pure trehalose because marginally self-aggregated HPO4(2-) ions established a strengthened hydrogen-bonding network with trehalose molecules. In contrast H2PO4(-) ions served only as plasticizers, resulting in a lower Tg of the mixtures than trehalose alone, creating large-sized ionic pockets, weakening interactions, and disrupting the original hydrogen-bonding network amongst trehalose molecules.

Effect of choline carboxylate ionic liquids on biological membranes.

Choline carboxylates, ChCm, with m=2-10 and choline oleate are known as biocompatible substances, yet their influence on biological membranes is not well-known, and the effect on human skin has not previously been investigated. The short chain choline carboxylates ChCm with m=2, 4, 6 act as hydrotropes, solubilizing hydrophobic compounds in aqueous solution, while the longer chain choline carboxylates ChCm with m=8, 10 and oleate are able to form micelles. In the present study, the cytotoxicity of choline carboxylates was tested using HeLa and SK-MEL-28 cells. The influence of these substances on liposomes prepared from dipalmitoylphosphatidylcholine (DPPC) was also evaluated to provide insights on membrane interactions. It was observed that the choline carboxylates with a chain length of m>8 distinctly influence the bilayer, while the shorter ones had minimal interaction with the liposomes.

Polymerization effect of electrolytes on hydrogen-bonding cryoprotectants: ion-dipole interactions between metal ions and glycerol.

Protectants which are cell membrane permeable, such as glycerol, have been used effectively in the cryopreservation field for a number of decades, for both slow cooling and vitrification applications. In the latter case, the glass transition temperature (Tg) of the vitrification composition is key to its application, dictating the ultimate storage conditions. It has been observed that the addition of some electrolytes to glycerol, such as MgCl2, could elevate the Tg of the mixture, thus potentially providing more storage condition flexibility. The microscopic mechanisms that give rise to the Tg-enhancing behavior of these electrolytes are not yet well understood. The current study focuses on molecular dynamics simulation of glycerol mixed with a variety of metal chlorides (i.e., NaCl, KCl, MgCl2, and CaCl2), covering a temperature range that spans both the liquid and glassy states. The characteristics of the ion-dipole interactions between metal cations and hydroxyl groups of glycerol were analyzed. The interruption of the original hydrogen-bonding network among glycerol molecules by the addition of ions was also investigated in the context of hydrogen-bonding quantity and lifetime. Divalent metal cations were found to significantly increase the Tg by strengthening the interacting network in the electrolyte/glycerol mixture via strong cation-dipole attractions. In contrast, monovalent cations increased the Tg insignificantly, as the cation-dipole attraction was only slightly stronger than the original hydrogen-bonding network among glycerol molecules. The precursor of crystallization of NaCl and KCl was also observed in these compositions, potentially contributing to weak Tg-enhancing ability. The Tg-enhancing mechanisms elucidated in this study suggest a structure-enhancing role for divalent ions that could be of benefit in the design of protective formulations for biopreservation purposes.

Determination of the relaxation characteristics of sugar glasses embedded in microfiber substrates.

Recently there has been considerable interest in developing sugar glasses that enable storage of biologics without refrigeration. Microfiber filter papers are good substrates for drying biologics in the presence of sugar glass-formers, providing for an even distribution of samples and an enhanced surface area for drying, but the opaqueness prevents macroscopic observation of the sample and can introduce complexities that impede physical characterization. Because drying kinetics and processing conditions can impact the relaxation dynamics (e.g., α- and ß-relaxation), which can influence the efficacy of the glass as a stabilizer, methods are needed that can enable a determination of relaxation phenomena of sugar glasses in such complex environments. In this study we present a method which provides verification of the absence of crystallinity following drying on glass fiber filter paper and also enables the determination of relaxation characteristics of amorphous sugar compositions embedded within these filter substrates. Using material pockets to contain the sugar glass-embedded microfiber paper, the α-relaxation temperature, Tα, was determined as a function of the water content in trehalose and sucrose samples using Dynamic Mechanical Analysis (DMA). Results were verified by comparison with previous calorimetric and spectroscopic studies. The data also demonstrated the plasticizing effects of water, as Tα was shown to correlate with water content via a Gordon-Taylor-like relationship. Our findings validate a new approach for determining the relaxation characteristics of microfiber embedded sugar glasses, and offer new insights into the relaxation characteristics of glasses prepared by microwave-assisted drying on filter papers.

Dynamic and thermodynamic characteristics associated with the glass transition of amorphous trehalose-water mixtures.

The glass transition temperature Tg of biopreservative formulations is important for predicting the long-term storage of biological specimens. As a complementary tool to thermal analysis techniques, which are the mainstay for determining Tg, molecular dynamics simulations have been successfully applied to predict the Tg of several protectants and their mixtures with water. These molecular analyses, however, rarely focused on the glass transition behavior of aqueous trehalose solutions, a subject that has attracted wide scientific attention via experimental approaches. Important behavior, such as hydrogen-bonding dynamics and self-aggregation has yet to be explored in detail, particularly below, or in the vicinity of, Tg. Using molecular dynamics simulations of several dynamic and thermodynamic properties, this study reproduced the supplemented phase diagram of trehalose-water mixtures (i.e., Tg as a function of the solution composition) based on experimental data. The structure and dynamics of the hydrogen-bonding network in the trehalose-water systems were also analyzed. The hydrogen-bonding lifetime was determined to be an order of magnitude higher in the glassy state than in the liquid state, while the constitution of the hydrogen-bonding network exhibited no noticeable change through the glass transition. It was also found that trehalose molecules preferred to form small, scattered clusters above Tg, but self-aggregation was substantially increased below Tg. The average cluster size in the glassy state was observed to be dependent on the trehalose concentration. Our findings provided insights into the glass transition characteristics of aqueous trehalose solutions as they relate to biopreservation.

Application of the Kwei equation to model the Tg behavior of binary blends of sugars and salts.

Vitrification of sugar-based solutions plays an important role in cryopreservation, lyophilization, and the emerging field of anhydrous preservation. An understanding of the glass transition characteristics of such formulations is essential for determining an appropriate storage temperature to ensure an extended shelf life of vitrified products. To better understand the effect of salts on the glass transition temperature (T(g)) of glass-forming sugars, we investigated several data-fitting models (Fox, Gordon-Taylor and Kwei) for sugar-salt formulations using data from the literature, as well as new data generated on blends of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA. The Kwei equation, which has a specific parameter characterizing intermolecular interactions, provides good fits to the T(g) data for sugar-salt blends, and complements other commonly used models that are frequently used to model T(g) data.

Interaction of choline salts with artificial biological membranes: DSC studies elucidating cellular interactions.

To better understand the relationship between the relative cytotoxicity of diluted ionic liquids and their specific interaction with biological membranes, the thermotropic behavior of model lipid membrane systems formulated in a series of choline based organic salts was investigated. Unilamellar vesicles prepared from dipalmitoylphosphatidylcholine were exposed to a series of choline phosphate salts at a concentration of 10mM at pH7.40, and the gel to liquid-crystalline state transition was examined using differential scanning calorimetry. The choline salts that were observed to have a low relative toxicity in previous studies induced minimal changes in the lipid phase transition behavior of these model membranes. In contrast, the salts choline bis(2,4,4-trimethylpentyl)phosphinate and choline bis(2-ethylhexyl)phosphate, both of which were observed to have high relative toxicity, caused distinct disruptions in the lipid phase transition behavior, consistent with penetration of the salts into the acyl chains of the phospholipids. choline bis(2,4,4-trimethylpentyl)phosphinate reduced the Tm and enthalpy of the main transition of dipalmitoylphosphatidylcholine while choline bis(2-ethylhexyl)phosphate induced the equilibration of alternate phases.

Advancing microwave technology for dehydration processing of biologics.

Our prior work has shown that microwave processing can be effective as a method for dehydrating cell-based suspensions in preparation for anhydrous storage, yielding homogenous samples with predictable and reproducible drying times. In the current work an optimized microwave-based drying process was developed that expands upon this previous proof-of-concept. Utilization of a commercial microwave (CEM SAM 255, Matthews, NC) enabled continuous drying at variable low power settings. A new turntable was manufactured from Ultra High Molecular Weight Polyethylene (UHMW-PE; Grainger, Lake Forest, IL) to provide for drying of up to 12 samples at a time. The new process enabled rapid and simultaneous drying of multiple samples in containment devices suitable for long-term storage and aseptic rehydration of the sample. To determine sample repeatability and consistency of drying within the microwave cavity, a concentration series of aqueous trehalose solutions were dried for specific intervals and water content assessed using Karl Fischer Titration at the end of each processing period. Samples were dried on Whatman S-14 conjugate release filters (Whatman, Maidestone, UK), a glass fiber membrane used currently in clinical laboratories. The filters were cut to size for use in a 13 mm Swinnex(®) syringe filter holder (Millipore(™), Billerica, MA). Samples of 40 µL volume could be dehydrated to the equilibrium moisture content by continuous processing at 20% with excellent sample-to-sample repeatability. The microwave-assisted procedure enabled high throughput, repeatable drying of multiple samples, in a manner easily adaptable for drying a wide array of biological samples. Depending on the tolerance for sample heating, the drying time can be altered by changing the power level of the microwave unit.

In vitro assessment of choline dihydrogen phosphate (CDHP) as a vehicle for recombinant human interleukin-2 (rhIL-2).

Choline dihydrogen phosphate (CDHP) is an ionic liquid reported to increase thermal stability of model proteins. The current work investigated CDHP effect on structural integrity and biological activity of recombinant human interleukin-2 (rhIL-2), a therapeutic protein used for treating advanced melanoma. In vitro CDHP biocompatibility was also evaluated using primary cell cultures, or B16-F10 cell line, chronically exposed to the ionic liquid. Formulation of rhIL-2 in an aqueous 680mM CDHP pH 7.4 solution resulted in a 12.5°C increase in the Tm of rhIL-2 compared to a basic buffer formulation, and provided conformational rhIL-2 stabilization when the solution was heated to 23.3°C above the Tm. CDHP solutions (≤80mM), exhibited no cytotoxic activity toward primary splenocytes or B16-F10 cells in culture. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30mM CDHP aqueous solution with NaHCO3 (pH≥7.2) compared to controls without CDHP. While increased Tm is associated with a diminished rhIL-2 biological activity, the therapeutic protein remains structurally intact and functional.