Functional activities of isolated lymphocytes following drying by sublimation of ice in vacuo: I. Rosette formation,stimulation by plant lectins (mitogens), and the mixed lymphocyte reaction

The following activities of isolated human lymphocytes were used for evaluating the effects of freezing and thawing and freeze-drying and rehydration on these cells: (a) spontaneous rosette formation, (b) responses to plant lectins (mitogens), and (c) the one-way mixed lymphocyte reaction. The successes achieved in drying of isolated lymphocytes by sublimation of ice in vacuo and rehydration with water with retention of the functions above, all of which appear to require living cells, were dependent upon a freeze-drying apparatus of unique design and the ability to freeze-dry suspending media containing dimethylsulfoxide. Best results were obtained when lymphocytes were: (a) isolated from blood collected in citrate-phosphate-dextrose (CPD); (b) suspended in Roswell Park Memorial Institute Medium-1640 (RPMI-1640) in sufficient amount to make 100%, 20% fetal calf serum, 8% serum albumin, 5% dimethylsulfoxide, and 1% L-glutamine; (c) cooled at approximately 1 °C/min from +4 to ?25 °C and approximately 5 °C/min from ?25 to ?70 °C, and (d) rehydrated at low temperatures.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

The molecular nature of damage by oxygen to freeze-dried Escherichia coli

The damage occurring in freeze-dried bacteria exposed to oxygen is mainly in the bacterial membrane and involves the DNA-initiation complex. This injury occurs in two stages: The primary damage is due to the freeze-drying itself, and is repaired upon reconstitution of bacteria and their subsequent incubation in nutrient broth. The repair process requires protein synthesis. In the next step, the exposure of freezedried bacteria to oxygen, the injury becomes irreversible and the bacteria “die.” The lethal effects of oxygen can be counteracted by either engaging the initiation sites on the membrane (e.g., by colicin E₁), or by arresting the activity of the initiation complex. Such arrest occurs in ts-mutants at nonpermissive temperature, or when appropriate mutants are starved of amino acids or thymine.After freeze drying and storage in vacuo, the reconstituted bacteria grow in filaments and synthetize DNA multifocally; they do not form septa and do not divide. After exposure to oxygen of the freeze-dried bacteria the ability to initiate DNA synthesis is lost.It is assumed therefore that oxygen specifically acts on the initiation complex in the bacterial membrane of the freeze-dried bacteria. When an external DNA synthesis control is provided, e.g., by infection with phage the productivity of the freeze-dried bacteria exposed to oxygen is the same as of the lyophilized controls held in vacuo.     

Phase diagram relationships in cryobiology

The reactions which occur during freezing in biological systems employing DMSO as a cryoprotective agent may well involve information given by a near equilibrium ternary H₂O-DMSO-NaCl phase diagram. The initial freezing point depressions for solutions with three different DMSO-NaCl initial ratios (R) have been determined over the onefold surface of ice saturation. DMSO has been shown to be more effective in reducing NaCl concentration in the residual liquid than had been previously predicted. The temperature and the fraction solid which must be reached for the occurrence of second phase coprecipitation with ice have been shown to be a strong function of initial R value. Ternary invariant reactions have been identified at ?35 °C, and tentatively identified at ?115 and ?105 °C for solutions having DMSO/NaCl ratios of R = 9, 5, and 1, respectively. Metastable nonequilibrium phase formation has been observed for slow cooling of a solution with R = 1. This metastable condition results in different phase relationships upon thawing than upon the initial freezing. By quenching the system after partial rewarming, it has been demonstrated that this metastable condition can be eliminated.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl₂ and CaCl₂). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl₂ and 0.125 M CaCl₂ compared to sea water. A combination of treatments with trehalose and MgCl₂ was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl₂/CaCl₂ (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.     

Development of a bladder instillation of the indoloquinone anticancer agent EO-9 using tert-butyl alcohol as lyophilization vehicle

The purpose of this research was to develop a stable bladder instillation of EO-9 for the treatment of superficial bladder cancer. First, stability and dissolution studies were performed. Subsequently, the freeze-drying process was optimized by determination of the freeze-drying characteristics of the selected cosolvent/water system and differential scanning calorimetry analysis of the formulation solution. Furthermore, the influence of the freeze-drying process on crystallinity and morphology of the freeze-dried product was determined with x-ray diffraction analysis and scanning electron microscopy, respectively. Subsequently, a reconstitution solution was developed. This study revealed that tert-butyl alcohol (TBA) can be used to both dramatically improve the solubility and stability of EO-9 and to shorten the freeze-drying cycle by increasing the sublimation rate. During freeze drying, 3 TBA crystals were found: TBA hydrate-ice crystals, crystals of TBA hydrate, and a third crystal, probably composed of TBA hydrate crystals containing ≈90% to 95% TBA. Furthermore, it was shown that crystallization of TBA hydrate was inhibited in the presence of both sodium bicarbonate (NaHCO₃) and mannitol. Addition of an annealing step resulted in a minor increase in the crystallinity of the freeze-dried product and formation of the δ-polymorph of mannitol. A stable bladder instillation was obtained after reconstitution of the freeze-dried product (containing 8 mg of EO-9, 20 mg of NaHCO₃, and 50 mg of mannitol per vial) to 20 mL with a reconstitution solution composed of propylene glycol/water for injection (WfI)/NaHCO₃/sodium edetate 60%/40%/2%/0.02% vol/vol/wt/wt, followed by dilution with Wfl to a final volume of 40 mL. Published: August 3, 2007     

Scanning Electron Microscopy of Heart Muscle Freeze-Dried from Dimethylsulfoxide for Simultaneous Demonstration of Cell Morphology and Microvascular Function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 ± 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Renaturation of triple helical polysaccharide lentinan in water-diluted dimethylsulfoxide solution

Triple helical lentinan, a β-(1→3)-d-glucan from Lentinus edodes, was denatured in dimethylsulfoxide (DMSO) into single random coils. The randomly coiled lentinan/DMSO solutions were diluted with pure water to w_H (the weight fraction of water in the mixed solvent) of 95%, and their intrinsic viscosity [η], weight-average molecular weight M_w, radius of gyration R_g, and hydrodynamic radius R_h were investigated at 25 °C after over 5-day storage. The [η] and M_w values, especially the conformation parameter ρ (≡R_g/R_h), of the renatured lentinan were close to those of the originally extracted one, suggesting that random lentinan chains in DMSO were reassembled into triple helical structures. Moreover, the renatured lentinan in 95% water/5% DMSO solution exhibited a unique behavior of triple helical glucans that shear modulus G′ decreased sharply at temperature from 8.4 °C to 13.3 °C with increasing temperature, which was ascribed to the intramolecular conformation transition from ordered triple helical I to disordered triple helical II. The AFM images gave was suggested intuitively evidence that the renatured lentinan mainly existed as rod-like chains, supporting that formation of triple helical structure. The optimal lentinan concentration for triple helical configuration formation was estimated to be over 0.04%. The time dependence of R_h and UV absorption of the water-diluted lentinan/DMSO solution with an indicator of azo dye of Congo red suggested that renaturation of triple helix was a very rapid process. Moreover, the blue-shift of UV-vis absorption spectra suggested that the dye molecules of Congo red were assembled into supramolecular structure in the hydrophobic cavity of the renatured triple helical lentinan. All the results showed that the triple helical structure formed once the randomly coiled lentinan/DMSO was diluted to the final water content of 95%.     

Scanning electron microscopy of heart muscle freeze-dried from dimethylsulfoxide for simultaneous demonstration of cell morphology and microvascular function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 +/- 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Heat and mass transfer scale-up issues during freeze drying: II. Control and characterization of the degree of supercooling

This study aims to investigate the effect of the ice nucleation temperature on the primary drying process using an ice fog technique for temperature-controlled nucleation. In order to facilitate scale up of the freeze-drying process, this research seeks to find a correlation of the product resistance and the degree of supercooling with the specific surface area of the product. Freeze-drying experiments were performed using 5% wt/vol solutions of sucrose, dextran, hydroxyethyl starch (HES), and mannitol. Temperature-controlled nucleation was achieved using the ice fog technique where cold nitrogen gas was introduced into the chamber to form an “ice fog”, there-by facilitating nucleation of samples at the temperature of interest. Manometric temperature measurement (MTM) was used during primary drying to evaluate the product resistance as a function of cake thickness. Specific surface areas (SSA) of the freeze-dried cakes were determined. The ice fog technique was refined to successfully control the ice nucleation temperature of solutions within 1°C. A significant increase in product resistance was produced by a decrease in nucleation temperature. The SSA was found to increase with decreasing nucleation temperature, and the product resistance increased with increasing SSA. The ice fog technique can be refined into a viable method for nucleation temperature control. The SSA of the product correlates well with the degree of supercooling and with the resistance of the product to mass transfer (ie, flow of water vapor through the dry layer). Using this correlation and SSA measurements, one could predict scaleup drying differences and accordingly alter the freeze-drying process so as to bring about equivalence of product temperature history during lyophilization.     

Synthesis and mass spectra of esters of branched chain fatty acids

The Wittig reaction between alkylidene triphenylphosphoranes [R — CH = P(C₆H₅)₃, where R varied from H to n — C₁₇H₃₅] and methyl 12-oxooctadecanoate or methyl 10-oxohexadecanoate in dimethylformamide (DMF) has been employed in the synthesis of a partial homologous series of esters of branched chain fatty acids in high yields. The effect of various ratios of reactants in both DMF and dimethylsulfoxide (DMSO) was investigated. Purification from triphenylphosphine oxide was readily accomplished by chromatography on a column of silicic acid-Celite impregnated with silver nitrate.     

Spatial Variation of Pressure in the Lyophilization Product Chamber Part 2: Experimental Measurements and Implications for Scale-up and Batch Uniformity

Product temperature during the primary drying step of freeze-drying is controlled by a set point chamber pressure and shelf temperature. However, recent computational modeling suggests a possible variation in local chamber pressure. The current work presents an experimental verification of the local chamber pressure gradients in a lab-scale freeze-dryer. Pressure differences between the center and the edges of a lab-scale freeze-dryer shelf were measured as a function of sublimation flux and clearance between the sublimation front and the shelf above. A modest 3-mTorr difference in pressure was observed as the sublimation flux was doubled from 0.5 to 1.0 kg·h^?1·m^?2 at a clearance of 2.6 cm. Further, at a constant sublimation flux of 1.0 kg·h^?1·m^?2, an 8-fold increase in the pressure drop was observed across the shelf as the clearance was decreased from 4 to 1.6 cm. Scale-up of the pressure variation from lab- to a manufacturing-scale freeze-dryer predicted an increased uniformity in drying rates across the batch for two frequently used pharmaceutical excipients (mannitol and sucrose at 5% w/w). However, at an atypical condition of shelf temperature of +10°C and chamber pressure of 50 mTorr, the product temperature in the center vials was calculated to be a degree higher than the edge vial for a low resistance product, thus reversing the typical edge and center vial behavior. Thus, the effect of local pressure variation is more significant at the manufacturing-scale than at a lab-scale and accounting for the contribution of variations in the local chamber pressures can improve success in scale-up.     

The effect of dimethylsulfoxide on the mutagenicity of the hair dye p-phenylenediamine

Published data on the mutagenicity of the oxidative hair dye intermediate p-phenylenediamine in the Ames test are conflicting. The work reported here resolves the discrepancies, showing that the activity of p-phenylenediamine in the Ames Salmonella/microsome test is markedly influenced by the use of dimethylsulfoxide as solvent, and by the age of the solution prior to plating. Thus, aqueous solutions of p-phenylenediamine are non-mutagenic; fresh solutions in DMSO are equally non-mutagenic, but become highly active on standing at room temperature for 4 h. These results suggest the need for caution in the choice of solvents and tight controls in the execution of the test.     

Thermal stability of freeze-dried mammalian interferons. Analysis of freeze-drying conditions and accelerated storage tests for murine interferon.

Conditions of Interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions.The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 m sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacuo with a starting temperature of ?30 °C to a final residual moisture of about 3%. The final product, reference reagent G002-904-511, was stable throughout the course of the LNS test. From an extensive MIS test, this reference interferon was predicted to lose 1000 units of activity in 110 years at 4 °C and 1000 units in 100 days at 37 °C. After 6 years of storage at 37 °C when the predicted residual activity would be about 20% of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test.     

Thallium(III) chloride in organic solvents: Synthesis, solutions and solvates. The crystal structures of trichlorobis(dimethylsulfoxide)thallium(III) and tribromobis(dimethylsulfoxide)thallium(III)

The synthesis of thallium(III) chloride and bromide was performed in solution by chlorination and bromination, respectively, of the suspensions of the corresponding thallium(I) halides in acetonitrile. Crystalline compounds TlX₃(CH₃CN)₂ (X = Cl⁻, Br⁻) were prepared from the acetonitrile solutions. Thallium(III) chloride and bromide in dimethylsulfoxide solution were obtained by dissolving the corresponding solid compounds TlX₃(CH₃CN)₂ (Cl⁻, Br⁻) in DMSO. Both acetonitrile and dimethylsulfoxide solutions of thallium(III) chloride were studied by UV-Vis and ²⁰⁵Tl NMR spectroscopy. The UV-Vis study of the TlCl₃-CH₃CN system showed presence of at least two thallium(III) chloride species. Only one signal arising from the thallium(III) species was, however, detected by the ²⁰⁵Tl NMR in the solution because of the fast chemical exchange. The ²⁰⁵Tl NMR study of thallium(III) chloride in dimethylsulfoxide showed three separate signals assigned to the solvated , TlCl₃ and species. The crystalline compounds of trichlorobis(dimethylsulfoxide)thallium(III) and tribromobis(dimethylsulfoxide)thallium(III) were prepared and their crystal structures were solved by single-crystal X-ray analysis. The thallium atom in the complexes has a trigonal bipyramidal environment built by three halide ions occupying equatorial positions of the polyhedron and two oxygen atoms of the DMSO molecules in the apical positions.     

Cryopreservation of isolated rat hepatocytes

Summary Isolated parenchymal hepatocytes from adult rats were frozen in media containing 10% glycerol, 10% dimethylsulfoxide (DMSO), or 20% DMSO. Three microsome-associated functions were compared in nonfrozen cells and cells frozen in each of the above cryoprotectant solutions. Freezing in DMSO maintains cytochromes P-450 and b₅ and NADPH-cytochrome C reductase at levels nearer to control values than does freezing in glycerol. Cells frozen and subsequently thawed and cultured for 24 h lose a greater amount of cytochrome P-450 than do nonfrozen cultured cells. The levels of cytochrome b₅ and reductase in frozen-thawed cells remain close to control values. Cell viability (trypan blue dye exclusion and percentage of attached cells) after freezing is maintained better using DMSO as a cryoprotectant. Dimethylsulfoxide protects the hepatocytes from freeze-induced damage to the extent that many viable cells attach to collagen-coated petri dishes, survive for at least 24 h, and still maintain significant levels of enzymes of importance to drug and carcinogen metabolism. This work was supported by Grant CA-30241 from the National Institutes of Health, Bethesda, Maryland.     

Mutagenic activity of platinum and ruthenium complexes

cis-Dichlorodiammineplatinum(II) [cis-PtCl₂(NH₃)₂] and dichlorotetrakis (dimethylsulfoxide) ruthenium(II) [RuCl₂(DMSO)₄] have been tested as mutagens for strains of Salmonella typhimurium carrying the hisG46 missense mutation. Their activity, which has been compared with the activity of mitomycin C, depends on the presence in the test bacteria of the pKM101 plasmid and is affected in various ways by the function of the excision repair system. More precisely, mitomycin C is mutagenic only for strains with an intact uvr system. cisPtCl₂(NH₃)₂ and RuCl₂(DMSO)₄ are mutagens both for uvrB and uvr⁺ strains, but cis-PtCl₂(NH₃)₂ is more active on the latter, while the converse is true for RuCl₂(DMSO)₄. It seems, therefore, that each drug interacts with DNA by a different mechanism.     

Isolation of a wheat cell line with altered membrane properties

A spontaneous dimethylsulfoxide (DMSO)-tolerant cell line was isolated from a cell culture of wheat (Triticum monococcum L.). The tolerant cells were able to grow in the presence of 4% DMSO. Cells formed from protoplasts of the tolerant line required DMSO for division in culture medium of high osmotic value.