The response of multilamellar liposomes to freezing and thawing

The use of liposomes as a model system for investigating the mechanism of freezing injury was investigated. Modification of the liposome phospholipid and cholesterol content allows a correlation to be made between the composition of a membrane system and its response to the stresses of freezing and thawing. The data on phase transitions are contradictory in the sense that liposomes become more sensitive to freezing injury following treatments which both increase or decrease phase transition temperature. In contrast the effect of cholesterol in sensitizing membranes to the stresses of freezing and thawing appears to be more fundamental. Direct cryomicroscope observations of liposomes during slow cooling indicate that they are osmotically active at low temperatures and upon thawing morphological alterations to the membranes occur. The response of liposomes following cooling at a range of rates to ?196 °C and the effects of cryoprotective additives are similar to those observed with many cell types. These results indicate that liposomes are a valid model for investigating the biochemistry of membrane damage induced by the stresses of freezing and thawing.     

Modeling conductive heat transfer during high-pressure thawing processes: determination of latent heat as a function of pressure

A numerical heat transfer model for predicting product temperature profiles during high-pressure thawing processes was recently proposed by the authors. In the present work, the predictive capacity of the model was considerably improved by taking into account the pressure dependence of the latent heat of the product that was used (Tylose). The effect of pressure on the latent heat of Tylose was experimentally determined by a series of freezing experiments conducted at different pressure levels. By combining a numerical heat transfer model for freezing processes with a least sum of squares optimization procedure, the corresponding latent heat at each pressure level was estimated, and the obtained pressure relation was incorporated in the original high-pressure thawing model. Excellent agreement with the experimental temperature profiles for both high-pressure freezing and thawing was observed.     

Involvement of two specific causes of cell mortality in freeze-thaw cycles with freezing to -196 degrees C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to -196 degrees C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than -70 degrees C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.     

Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

Water transport and cell survival in cryobiological procedures.

Living cells may be cooled to 77 K (liquid nitrogen) either to destroy them selectively or to store them for long periods. Water transport across the cell membranes during freezing and thawing is a primary factor determining whether the cells survive. These water movements are controlled by phase changes both intracellular and extracellular and by other factors such as the nature of any cryoprotective agent present, and the rates of cooling and thawing. The relation between cooling procedure, water transport and cell survival is discussed. In particular, the crucial r?le of dilution shock is emphasized: this is the damage to cells induced during the dilution that occurs both as ice melts during rewarming and when any cryoprotective additives are removed after thawing. Apart from the usefulness of understanding these processes for maximizing preservation or controlling selective destruction, the diverse responses of cells to different combinations of water transport and temperature changes appear likely to provide basic information on the properties of cell membranes.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.     

Heat shock protects germinating conidiospores of Neurospora crassa against freezing injury.

Germinating conidiospores of Neurospora crassa that were exposed to 45 degrees C, a temperature that induces a heat shock response, were protected from injury caused by freezing in liquid nitrogen and subsequent thawing at 0 degrees C. Whereas up to 90% of the control spores were killed by this freezing and slow thawing, a prior heat shock increased cell survival four- to fivefold. Survival was determined by three assays: the extent of spore germination in liquid medium, the number of colonies that grew on solid medium, and dry-weight accumulation during exponential growth in liquid culture. The heat shock-induced protection against freezing injury was transient. Spores transferred to normal growth temperature after exposure to heat shock and before freezing lost the heat shock-induced protection within 30 min. Spores subjected to freezing and thawing stress synthesized small amounts of the heat shock proteins that are synthesized in large quantities by cells exposed to 45 degrees C. Pulse-labeling studies demonstrated that neither chilling the spores to 10 degrees C or 0 degrees C in the absence of freezing nor warming the spores from 0 degrees C to 30 degrees C induced heat shock protein synthesis. The presence of the protein synthesis inhibitor cycloheximide during spore exposure to 45 degrees C did not abolish the protection against freezing injury induced by heat shock. Treatment of the cells with cycloheximide before freezing, without exposure to heat shock, itself increased spore survival.     

Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: observations with experimental cryomicroscopy.

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10 degrees C retained fluorescence into the post-thaw temperature range of 9-24 degrees C (mean +/- SEM; 13.25 +/- 0.91 degrees C), whereas cells cooled to -20 degrees C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91 degrees C). Sperm cooled to 5 degrees C, but not frozen, retained fluorescence during rewarming up to 20-30 degrees C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.     

利用飞秒激光研究微液滴的冻结相变特性

对微液滴冻结行为的认识在低温生物学、分析化学等方面具有重要意义.引入飞秒激光实验手段研究液滴及微量生物材料(蛋白)的冻结相变特性.实验考察了样品在多次冻结过程中荧光光谱的变化规律,结果表明:生物材料与非生物材料在冻结及复温过程中的荧光光谱变化趋势存在差异,非生物试剂在冻结过程中光谱下降,经历复温后,其光谱可回复到初始状态;而蛋白在冻结过程中光谱上升,经历复温后,由于降温/升温过程对其造成的不可逆损伤,光谱无法回复到初始状态.基于此提出了用以评估生物样品活性的非接触式飞秒激光测量方法.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

The effects of different thawing temperatures on morphology and collagen metabolism of -20 degrees C dealt normal human fibroblast

The purpose of present study is to investigate the effects of two different thawing temperatures on normal human fibroblast which dealt with -20 degrees C, hoping to provide a clue for further study in reducing excessive collagen formation after cryotherapy on skin diseases in vitro, as well as in differentiation disorders. In order to elucidate its action mechanism, a programmable freezing device was developed to apply freezing temperatures on cell cultures. The effects of two different thawing temperatures on frozen fibroblast proliferation, viability, collagen synthesis and alpha smooth muscle actin (alpha-SMA) expressing were investigated. We found that compared with 37 degrees C, thawing with 20 degrees C yielded same motility. But there are significant differences in terms of the alpha-SMA expression (P<0.05) of fibroblast and collagen I, III synthesis (P<0.01) between two groups after 72h. The results suggest that comparing with slow thawing; rapid thawing cannot only keep the same cell's damage, but also can modify collagen synthesis and differentiation of fibroblasts. It may be more suitable for the cryosurgical treatment of keloids and benign skin diseases.     

Reversible Photoinhibition in Antarctic Moss during Freezing and Thawing

Tolerance of antarctic moss to freezing and thawing stress was investigated using chlorophyll a fluorescence. Freezing in darkness caused reductions in Fv/Fm (ratio of variable to maximum fluorescence) and Fo (initial fluorescence) that were reversible upon thawing. Reductions in Fv/Fm and Fo during freezing in darkness indicate a reduction in the potential efficiency of photosystem II that may be due to conformational changes in pigment-protein complexes due to desiccation associated with freezing. The absorption of light during freezing further reduced Fv/Fm and Fo but was also reversible. Using dithiothreitol (DTT), which inhibits the formation of the carotenoid zeaxanthin, we found reduced flurorescence quenching during freezing and reduced concentrations of zeaxanthin and antheraxanthin after freezing in DTT-treated moss. Reduced concentrations of zeaxanthin and antheraxanthin in DTT-treated moss were partially associated with reductions in nonphotochemical fluorescence quenching. The reversible photoinhibition observed in antarctic moss during freezing indicates the existence of processes that protect from photoinhibitory damage in environments where freezing temperatures occur in conjunction with high solar radiation levels. These processes may limit the need for repair cycles that require temperatures favorable for enzyme activity.     

冻融过程对荒漠短命植物种子萌发的影响

荒漠区冻融交替显著改变土壤温度和水分条件,并进一步影响荒漠植物种子萌发.为解析荒漠土壤冻融过程对植物种子萌发的影响,本研究以古尔班通古特沙漠4种典型短命植物[东方旱麦草(Eremopyrum orientale)、卵果鹤虱(Lappula patula)、尖喙拢牛儿苗(Erodiumoxyrrhychum)和条叶庭荠(...     

Improved and reproducible cell viability in the superflash freezing method using an automatic thawing apparatus

Cell cryopreservation stops the biological activity of cells by placing them in the frozen state, and can be used to preserve cells without subculturing, which can cause contamination and genetic drift. However, the freezing process used in cryopreservation can injure or damage the cells due to the cytotoxicity of cryoprotecting agents (CPAs). We have previously reported a CPA-free cryopreservation method based on inkjet technology. In this method, the vitrified cells were exposed to the room temperature atmosphere during the transport of the cells using tweezers, which caused devitrification due to the increased temperature and often lowered the cell viability. In the present study, we developed an automatic thawing apparatus that transports the vitrified cells rapidly into a prewarmed medium using a spring hinge. Observations with a high-speed camera revealed that the spring hinge drops the cells into the prewarmed medium within 20 ms. All heat-transfer simulations for the apparatuses with different designs and rotation speeds showed that the cells remained below the glass-transition temperature during the transport. Finally, the apparatus was evaluated using mouse fibroblast 3T3 cells. The cell viability was improved and its reproducibility was enhanced using this apparatus. The results indicate that the combination of superflash freezing with the rapid thawing process represents a promising approach to circumvent the problems typically associated with the addition of CPAs.     

Reduction of photosynthetic apparatus plays a key role in survival of the microalga Haematococcus pluvialis (Chlorophyceae) at freezing temperatures

The microalga Haematococcus pluvialis is a biotechnologically important microorganism producing a ketocarotenoid astaxanthin. Haematococcus exists either as metabolically active vegetative cells with a high chlorophyll content or astaxanthin-rich haematocysts (aplanospores). This microalga featuring outstanding tolerance to a wide range of adverse conditions is a highly suitable model for studies of freezing tolerance in phototrophs. The retention of H. pluvialis cell viability after freezing–thawing is ascribed to elevated antioxidant enzyme activity and high ketocarotenoid content. However, we report that only haematocysts characterized by a lower photosynthetic activity were resistant to freezing–thawing even without cryoprotectant addition. The key factors of haematocyst freezing tolerance were assumed to be a low water content, rigid cell walls, reduction of the membranous structures, photosynthesis downregulation, and low chlorophyll content. Collectively, viability of Haematoccus after freezing–thawing can be improved by forcing the transition of vegetative cells to freeze-tolerant haematocysts before freezing.

     

川西亚高山季节性冻土期针叶林主要树种叶片和细根的生态生理特征

川西亚高山森林存在明显的季节性冻土现象, 该地区的土壤经历着初冬冻融、深冬冻结、早春冻融等过程, 同时,该区域冬季受气候变化的影响强烈。为了全面地认识亚高山森林的生态过程, 该研究以川西亚高山针叶林两种主要树种——岷江冷杉(Abies fargesii var. faxoniana)和云杉(Picea asperata)为材料, 研究其叶片及细根内丙二醛含量、渗透调节物质的含量、组织含水量、过氧化物酶活性以及硝酸还原酶活性在季节性冻土期的变化, 同时还比较了冻土期和冻融期细根的比根长, 比表面积, 直径以及组织密度的变化。研究结果显示: 在季节性冻土期, 土壤温度昼夜波动幅度小于空气温度波动幅度, 细根却表现出更强的过氧化物酶活性以及更高的渗透调节物质含量, 说明细根较叶片对季节性冻土更为敏感。与冻结期相比, 冻融期土壤温度、空气温度以及空气相对湿度昼夜波动幅度增加, 促使云杉叶片可溶性糖含量以及两针叶树种叶片内过氧化物酶活性、脯氨酸含量显著增加, 而细根的组织含水量显著降低, 脯氨酸、可溶性蛋白质及可溶性糖含量均显著增加, 表明冻融期对两针叶树种的影响较冻结期更为强烈。岷江冷杉和云杉的过氧化物酶活性及渗透调节物质含量具有相同的变化趋势, 但叶片和细根的膜脂过氧化程度及酶活性变化并不一致, 就岷江冷杉而言, 细根的丙二醛含量显著增加, 而叶片、细根的硝酸还原酶活性均显著降低, 云杉仅叶片的丙二醛含量发生变化, 且显著降低, 说明云杉更能忍耐冻融循环造成的胁迫。研究还发现细根形态在季节性冻土期无显著变化。     

ATP involvement in plant tissue responses to low temperature

The effects of chilling and freezing temperature on membrane permeability and ATP content were studied in the leaves of cucumber ( Cucumis sativus L.) and winter rape ( Brassica napus L. var. oleifera L.) leaves, grown at different temperatures. In the winter rape leaves, the endogenous ATP content was modified by application of dinitrophenol (DNP) solutions of different concentrations. The low temperature-induced changes in membrane permeability (as monitored by the conductivity method) were found to be associated with ATP decrease, both in the chilling-sensitive and chilling-resistant (subjected to freezing) plants. In tissues showing reversible injuries, changes in ATP content preceded those in membrane permeability and the adenylate energy charge was affected slightly. In tissues showing irreversible membrane damage, the ATP content was always below 0.4 μmol (g dry weight)⁻¹ and the adenylate energy charge was near 0.5. DNP treatment increased freezing sensitivity of winter rape leaves. In the cold-hardened winter rape leaves, however, freezing and thawing did not significantly affect ATP content or the energy charge, although the specimen showed a rather large increase in membrane permeability. In these leaves ATP content recovered about 20 h after a freezing and thawing treatment. It is proposed that a decrease in ATP supply might be the primary reason for the membrane leakiness at low temperature, both in chilling-sensitive and chilling-resistant (subjected to freezing) plants. The conclusion is, however, not true for the cold-acclimated, frostadapted cells.