The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol–acetamide–Ficoll–sucrose solution were cooled to −196 °C at rates ranging from 37 to 1827 °C/min between 20 and −120 °C, and for each cooling rate, warmed at rates ranging from 139 to 2950 °C/min between −70 and −35 °C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827 °C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.     

Vitrification of one-cell mouse embryos in cryotubes

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.     

Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

Characterizing the freezing behavior of liposomes as a tool to understand the cryopreservation procedures

Freezing behaviors of egg yolk l-α-phosphatidylcholine (EPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicles (LUV) were quantitatively characterized in relation to freezing temperatures, cooling rates, holding time, presence of sodium chloride and phospholipid phase transition temperature. Cooling of the EPC LUV showed an abrupt increase in leakage of the encapsulated carboxyfluorescein (CF) between −5 °C and −10 °C, which corresponded with the temperatures of the extraliposomal ice formation at around −7 °C. For the DPPC LUV, CF leakage started at −10 °C, close to the temperature of the extraliposomal ice formation; followed by a subsequent rapid increase in leakage between −10 °C and −25 °C. Scanning electron microscopy showed that both of these LUV were freeze-concentrated and aggregated at sub-freezing temperatures. We suggest that the formation of the extraliposomal ice and the decrease of the unfrozen fraction causes freeze-injury and leakage of the CF. The degree of leakage, however, differs between EPC LUV and DPPC LUV that inherently vary in their phospholipid phase transition temperatures. With increasing holding time, the EPC LUV were observed to have higher leakage when they were held at −15 °C compared to at −30 °C whilst leakage of the DPPC LUV was higher when holding at −40 °C than at −15 °C and −50 °C. At slow cooling rates, osmotic pressure across the bilayers may cause an additional stress to the EPC LUV. The present work elucidates freeze-injury mechanisms of the phospholipid bilayers through the liposomal model membranes.     

Differences in cold and drought tolerance of high arctic and sub-arctic populations of Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola)

The springtail Megaphorura arctica (Onychiuridae: Collembola) inhabits the arctic and sub-arctic parts of the northern hemisphere where it on a seasonal basis will be exposed to severe cold and desiccating conditions. In the present study we compared how traits of stress resistance differed between two populations of M. arctica that were collected at a high arctic site (Spitsbergen) and a sub-arctic site (Akureyri, Iceland) with contrasting thermal environments. In addition we investigated how cold and desiccation affected the phospholipid fatty acid composition of M. arctica from Spitsbergen. The springtails from Spitsbergen were the most cold tolerant and this was linked to an almost three times higher level of trehalose accumulation during cryoprotective dehydration (15% and 5% of tissue dry weight in the Spitsbergen and Iceland populations, respectively). Although cryoprotective dehydration is intimately related to desiccation stress it was shown that M. arctica had a higher mortality when dehydrated over ice (−10 or −20 °C) than when dehydrated at temperatures above 1 °C. Thus, survival was lower after exposure to −10 °C than after exposure to a relative humidity of 91.2% RH at +1 °C although both treatments led to the same level of dehydration. Exposure to both cold (−10 and −20 °C) and desiccation at +1 °C caused significant changes in the phospholipid fatty acid composition with some similarities. These changes included a decrease in average chain length of the fatty acids due primarily to an increase in the phospholipid fatty acids 16:0 and a decrease in 18:3 and 20:4ω6.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Response of cylindrospermopsin production and release in Aphanizomenon flos-aquae (Cyanobacteria) to varying light and temperature conditions

The influence of light and temperature on the cylindrospermopsin (CYN) production of two Aphanizomenon flos-aquae strains, isolated from North-eastern German lakes, was investigated with semi-continuously growing cultures. A light gradient from 10 to 60 μE m⁻² s⁻¹ in combination with temperatures of 16, 20, and 25 °C was tested.CYN concentrations varied by a maximum factor of 2.7 in strain 10E9 with a significant decrease with increasing temperature. Strain 22D11 showed less pronounced changes, i.e. by a factor of 1.6, and without clear relationship to temperature.Reaction patterns of CYN production to changing light intensities are different at different temperatures. In both strains CYN concentrations increase significantly at 20 °C between 10 and 60 μE m⁻² s⁻¹, whereas they decrease significantly at 25 °C in the same light gradient. The amount of synthesised CYN is not reflected by growth rates of the strains in a uniform manner. Nonetheless several temperature–light combinations which constitute physiological stress seem to trigger CYN production and particularly CYN release from cells. The lowest growth rate observed at 16 °C and 60 μE m⁻² s⁻¹ of strain 22D11 may reflect photoinhibition due to the lower temperature and related limited CO₂-fixation. Under these conditions, extracellular CYN concentrations increased to 58% of total CYN, while the share of extracellular CYN of all other light and temperature regimes was 11–26%. From the results and the experimental design we conclude an active release of the toxin into medium to be more likely than mere leakage from cells.     

Gut Colonization by an Ice Nucleation Active Bacterium,Erwinia(Pantoea)ananasReduces the Cold Hardiness of Mulberry Pyralid Larvae

To evaluate the suitability of using ice nucleation active (INA) bacteria for the biological control of insect pests, the supercooling point (SCP) of larvae of mulberry pyralid,Glyphodes duplicalis,and silkworm,Bombyx mori,ingesting INA strains ofErwinia(Pantoea)ananasandPseudomonas syringaewas determined. Mean SCP of the guts of silkworm larvae ingesting INA strains ofE. ananasranged from −2.5 to −2.8°C, being 5°C higher than that in control treatments. Similarly, mean SCP of mulberry pyralid larvae ingesting INA strain ofE. ananas,which can grow well in the gut, was −4.7°C at 3 days after treatment, being 6.5°C higher than that in control treatments. On the other hand, mean SCP of the larvae-ingesting INA strain ofP. syringae,which cannot grow in the gut, was −9.0°C at 3 days after treatment, rising by only 2.5°C higher than that in the control treatments. In addition, more than 80% of the larvae of mulberry pyralid ingesting the INA strain ofE. ananasfroze and eventually died when exposed to −6°C for 18 h, while only 36% of the larvae ingesting the INA strain ofP. syringae,or approximately 20% of the control larvae, froze and died. Thus, the gut colonization by INA strains ofE. ananasreduced remarkably the cold hardiness of the insects. These findings suggest that INA strains ofE. ananascould be effective as a potential biological control agent of insect pests.     

Synthesis, characterization and aqueous dispersion of dextran-g-poly(1,4-dioxan-2-one) copolymers

A novel biodegradable graft copolymer, dextran-g-poly(1,4-dioxan-2-one) (PODEX), was synthesized through the ring-opening polymerization (ROP) of 1,4-dioxan-2-one (PDO) onto a dextran backbone. Initially, dextran was silylated with 1,1,1,3,3,3-hexamethyldisilazane. The grafting from pathway was conducted with various (30–70) PDO/OH feed ratios to obtain PODEX copolymers with a various PPDO graft structures. Graft copolymers were characterized by FT-IR, ¹H and ¹³C NMR, DSC, TGA, and WAXD. Molecular weights of the PODEX copolymers (M_W: 94,700–117, 300 Da), glass transition temperature (−29 to −17 °C), melting temperature (82–100 °C), and crystallinity (32–40%) were increased with the content of PPDO. AFM observations revealed that polymeric micelles were spherical and uniform in morphology with around 30–58 nm diameter. Critical micelle concentration (CMC) of self-assembled system was significantly decreased from 3.2 to 1.09 mg/L with the increment of PPDO.     

Thermoregulation of water foraging wasps (Vespula vulgaris and Polistes dominulus)

A comparison of the thermoregulation of water foraging wasps (Vespula vulgaris, Polistes dominulus) under special consideration of ambient temperature and solar radiation was conducted. The body surface temperature of living and dead wasps was measured by infrared thermography under natural conditions in their environment without disturbing the insects’ behaviour. The body temperature of both of them was positively correlated with T_a and solar radiation. At moderate T_a (22–28 °C) the regression lines revealed mean thorax temperatures (T_th) of 35.5–37.5 °C in Vespula, and of 28.6–33.7 °C in Polistes. At high T_a (30–39 °C) T_th was 37.2–40.6 °C in Vespula and 37.0–40.8 °C in Polistes. The thorax temperature excess (T_th–T_a) increased at moderate T_a by 1.9 °C (Vespula) and 4.4 °C (Polistes) per kW⁻¹ m⁻². At high T_a it increased by 4.0 °C per kW⁻¹ m⁻² in both wasps. A comparison of the living water foraging Vespula and Polistes with dead wasps revealed a great difference in their thermoregulatory behaviour. At moderate T_a (22–28 °C) Vespula exhibited distinct endothermy in contrast to Polistes, which showed only a weak endothermic activity. At high T_a (30–39 °C) Vespula reduced their active heat production, and Polistes were always ectothermic. Both species exhibited an increasing cooling effort with increasing insolation and ambient temperature.     

Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts

Our objective was to study the effect of the concentration of ethylene glycol (EG) and dimethyl sulfoxide (Me₂SO) during vitrification on the development of porcine blastocysts. Vitrification was performed with 0.4 M sucrose and either a Me₂SO and EG mixture (15%, 16% and 17% v/v of each) or EG alone (40% v/v), using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 48 h and the survival and hatching rates were evaluated. Some vitrified and fresh embryos were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization to determine the proliferation index. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% of cryoprotectants, which displayed lower (P < 0.05) survival than fresh blastocysts. Vitrified and fresh blastocysts had a similar cell proliferation index (range: 75.8 ± 3.2 to 83.7 ± 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% of EG–Me₂SO. In conclusion, the concentration of EG–Me₂SO could be decreased to 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with similar results to those achieved with a medium containing 16% EG–Me₂SO.     

Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction

This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, −20, and −80 °C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 °C and lyophilized samples stored at +4 and −20 °C were the most stable. Samples stored as frozen aqueous solutions at −20 °C were the least stable.     

Clean conversion of cellulose into fermentable glucose

We studied the process of conversion of microcrystalline-cellulose into fermentable glucose in the formic acid reaction system using cross polarization/magic angle spinning ¹³C-nuclear magnetic resonance, X-ray diffraction and Fourier transform infrared spectroscopy. The results indicated that formic acid as an active agent was able to effectively penetrate into the interior space of the cellulose molecules, thus collapsing the rigid crystalline structure and allowing hydrolysis to occur easily in the amorphous zone as well as in the crystalline zone. The microcrystalline-cellulose was hydrolyzed using formic acid and 4% hydrochloric acid under mild conditions. The effects of hydrochloric acid concentration, the ratio of solid to liquid, temperature (55–75 °C) and retention time (0–9 h), and the concentration of glucose were analyzed. The hydrolysis velocities of microcrystalline-cellulose were 6.14 × 10^− 3 h^− 1 at 55 °C, 2.94 × 10^− 2 h^− 1 at 65 °C, and 6.84 × 10^− 2 h^− 1 at 75 °C. The degradation velocities of glucose were 0.01 h^− 1 at 55 °C, 0.14 h^− 1 at 65 °C, 0.34 h^− 1 at 75 °C. The activation energy of microcrystalline-cellulose hydrolysis was 105.61 kJ/mol, and the activation energy of glucose degradation was 131.37 kJ/mol.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

Enzymatic preparation of wheat bran xylooligosaccharides and their stability during pasteurization and autoclave sterilization at low pH

Xylooligosaccharides (XOS) were prepared from wheat bran insoluble dietary fiber (WBIDF) by treatment with commercial xylanase preparation Sunzymes. XOS, with a purity of 95% (w/w) and degree of polymerization of 2-7 and the ratio of arabinose to xylose of 0.27, was obtained with a yield of approximately 31.2% of WBIDF. Their stability was evaluated by comparing with that of commercial fructooligosaccharides (FOS) during pasteurization (60–100 °C, 30 min) and autoclave sterilization (121 °C, 1 kg/cm², 10–50 min) at pH 2.0–4.0. XOS was characterized by a high thermal stability during pasteurization at pH 2.5–4.0 and sterilization at pH 3.0–4.0. Even at pH 2.0, the remaining XOS reached 97.2% (w/w) and 84.2% (w/w) during pasteurization (100 °C, 30 min) and sterilization (50 min), respectively. Compared with FOS, XOS was strongly resistant to lower acidic conditions. The results revealed that XOS was considered to be more suitable for use as functional food ingredients.     

Renewal time and the impact of harmful algal blooms on the extensive mussel raft culture of the Iberian coastal upwelling system (SW Europe)

The extension and intensity of the upwelling season in the NW Iberian Peninsula (42°N–43°N) have decreased by 30 and 45% over the last 40 years, respectively. Accordingly, the renewal time (τ) of the Rías Baixas, four large coastal inlets where 15% of the World extraction of blue mussels occurs, has increased by 240%. We indirectly demonstrate here that the growing τ has caused the increasing occurrence of harmful microalgae in these embayments, dramatically affecting mussel raft cultivation. The equation D = 365(1 − exp(−τ/c₁)) explains 80% of the variability of the number of days per year that mussels cannot be extracted from the hanging ropes because of the occurrence of harmful microalgae (D). The coefficient c₁ = 37 ± 2 days indicates that an average τ over the upwelling season of >25 ± 1 or 50 ± 3 days reduce mussel extraction to only 50 or 25% of the year, respectively.     

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching −40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.     

Protease-catalyzed dipeptide synthesis from N-protected amino acid carbamoylmethyl esters and free amino acids in frozen aqueous solutions

The kinetically controlled synthesis of N-benzyloxycarbonyl (Z)-dipeptides was investigated by the use of free amino acids as nucleophiles and a cysteine protease papain as catalyst. The coupling efficiency was significantly improved by the combined use of the carbamoylmethyl (Cam) ester of a Z-amino acid as acyl donor and frozen aqueous solution (ice, −16 or −24 °C) as reaction medium. The yield of peptide synthesis became high when both P₁- and P^′₁-positions were occupied by small non-polar amino acids (Z-Gly-Gly-OH, 76%; Z-Gly-Ala-OH, 75%; Z-Ala-Ala-OH, 72%). Similar results were observed by the use of ficin as catalyst instead of papain. Furthermore, this strategy was applied to the papain-catalyzed incorporation of a d-configured amino acid such as d-alanine into the resulting peptides. Although the coupling in aqueous solution (30 °C) afforded the desired Z-dipeptides in low yields, the freezing of reaction medium reduced significantly unfavorable hydrolysis of the acyl donors, resulting in improvement of the coupling efficiency (Z-Gly-d-Ala-OH, 80%; Z-Ala-d-Ala-OH, 45%; Z-d-Ala-Ala-OH, 22%).