比较三种防冰晶法对切片保存效果的影响

目的 比较冰冻切片技术中几种防冰晶方法对切片保存效果的影响。方法 分别使用液氮骤冷组织、高渗透压脱水法、单纯OCT胶包埋等几种方法后行冰冻切片、HE染色后,分别于当天、1、3、6个月后比较其染色效果,选出最佳保存方法。结果 单纯OCT包埋法在1个月后染色明显变浅,冰晶数量最多,而在3、6个月后染色基本接近背底色,冰晶面积已占据近半个视野,组织结构破坏极其严重;高渗透压脱水法在1个月后染色深度及冰晶数量上无明显变化,但3、6个月后则明显变浅,冰晶数量明显增加;而低温骤冷法在染色后的几个月,染色深度及冰晶的数量均无明显的变化,结构基本维持染色当天的状态。结论 低温骤冷法是这三种防冰晶法中最利于保存切片的方法。     

Ultrastructural and thermocouple evaluation of rapid freezing techniques

The quality of freeze-fixation for electron microscopy is dependent upon the size of intracellular ice crystals. In the absence of cryoprotectants, ice crystal growth is thought to be related to the speed with which the specimen is cooled. The purpose of this study was to investigate the relationship between the cooling rate and ultrastructural preservation in commonly used freezing techniques. The techniques studied included immersion in stirred and unstirred forms of five quenching fluids: liquid nitrogen, isopentane, Freon 12, Freon 22, and propane. Also studied were freezing in a flowing stream of coolant using liquid nitrogen and liquid helium and freezing on a metal surface using cooper and mercury chilled to liquid nitrogen temperature. For each technique a cooling curve was obtained with a 0.360-mm thermocouple which was dropped into the quenching fluids or brought into contact with the metal surfaces. From oscilloscope tracings, the cooling rates were determined in degrees centigrade per second to −100 °C. To evaluate ultrastructural preservation 0.5-mm-thick slices of rat kidney were frozen by each of the techniques and dried in an all glass freeze-drier. The final evaluation was made from electron micrographs of the best morphological preservation yielded by each technique. The results indicate that the copper and mercury surfaces and propane gave the highest cooling rates and the best morphological preservation. The other techniques cooled at decreasing rates and correspondingly showed decreasing abilities to preserve ultrastructure. This work demonstrates that the preservation of cellular ultrastructure by freezing is dependent upon the cooling rate and that as the cooling rate is increased, ultrastructural preservation is enhanced.     

Control of ice recrystallization in liver tissues using small molecule carbohydrate derivatives

Minimizing ice recrystallization injury in tissues and organs has historically been sought using biological antifreeze proteins. However, the size of these compounds can limit permeation and their potential immunogenicity disqualifies them from use in several cryopreservation applications. Novel small molecule carbohydrate-derived ice recrystallization inhibitors (IRIs) are not subject to these constraints, and thus we sought to evaluate the ability of a highly active IRI to permeate liver tissue and control recrystallization. Rat liver tissue blocks (0.5 mm²) were incubated with the IRI for 6 h at 22 °C and subsequently plunged in liquid nitrogen. Ice crystals within the tissue were fixed using a formal acetic alcohol fixative as it was rewarmed from −80 °C to 22 °C over the course of 48 h. The untreated control demonstrated a gradient of increasing crystal size from the exterior to the interior region of the tissue; however, the IRI-treated condition had no such gradient and exhibited small crystals throughout. Threshold segmentation confirmed a significant reduction in the ice crystal size within the interior region of the IRI-treated condition, suggesting the IRI permeated throughout and effectively controlled recrystallization within the tissue.     

Large Ice Crystals in the Nucleus of Rapidly Frozen Liver Cells

Evidence in the literature shows that ice crystals that form in the nucleus of many rapidly cooled cells appear much larger than the ice crystals that form in the surrounding cytoplasm. We investigated the phenomenon in our laboratory using the techniques of freeze substitution and low temperature scanning electron microscopy on liver tissue frozen by liquid nitrogen plunge freezing. This method is estimated to cool the tissue at 1000°C/min. The results from these techniques show that the ice crystal sizes were statistically significantly larger in the nucleus than in the cytoplasm. It is our belief that this finding is important to cryobiology considering its potential role in the process of freezing and the mechanisms of damage during freezing of cells and tissues.     

Relationships between ice crystal size, water content and proton NMR relaxation times in cells

Biological specimens were frozen under controlled conditions. We questioned how the size of ice crystals, as measured in cryosectioned and cryoadsorbed sections of these biological specimens, relates to the water content and to the proton NMR relaxation times (T1 and T2) of the unfrozen specimens. The results permit the following conclusions: After rapid freezing in liquid propane cooled in a liquid nitrogen bath, the average size of ice crystals at distances of 150 microns or more from the surface of a particular tissue was always the same. Thus, the average size of the ice crystals was found to be characteristic of the type of biological tissue studied. Linear regression analysis showed average ice crystal size to have a significant correlation coefficient to T1 relaxation time and to water content. Specifically ice crystal size increased with T1 relaxation time and with water content. Multiple regression and path analysis demonstrated a positive correlation between the T1 relaxation time and the ice crystal size variation. Path analysis showed that both water content and T2 relaxation time were less directly correlated with ice crystal size. The findings from the path analysis and other observations show that the average size of ice crystals in subcellular compartments is best predicted by the proton T1 relaxation time. A working model is put forth to explain differences in ice crystal size observed between specimens enriched in globular or in parallel filamentous proteins.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.

Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at −78°. They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.

Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below −60°. Those cells at intermediate temperatures from −20° to −45°, were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at −30°. The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below −60° or into liquid nitrogen.

The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from −70° to −10° before rapid rewarming. There was little damage when samples were held at temperatures below −50° for 10 minutes or below −60° for 16 hours. No cavities were found in these cells. Above −45°, and especially at −30°, however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.     

Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

Ice Morphology: Fundamentals and Technological Applications in Foods

Freezing is the process of ice crystallization from supercooled water. Ice crystal morphology plays an important role in the textural and physical properties of frozen and frozen-thawed foods and in processes such as freeze drying, freeze concentration, and freeze texturization. Size and location of ice crystals are key in the quality of thawed tissue products. In ice cream, smaller ice crystals are preferred because large crystals results in an icy texture. In freeze drying, ice morphology influences the rate of sublimation and several morphological characteristics of the freeze-dried matrix as well as the biological activity of components (e.g., in pharmaceuticals). In freeze concentration, ice morphology influences the efficiency of separation of ice crystals from the concentrated solution. The cooling rate has been the most common variable controlling ice morphology in frozen and partly frozen systems. However, several new approaches show promise in controlling nucleation (consequently, ice morphology), among them are the use of ice nucleation agents, antifreeze proteins, ultrasound, and high pressure. This paper summarizes the fundamentals of freezing, methods of observation and measurement of ice morphology, and the role of ice morphology in technological applications.     

Glassy State and Cryopreservation of Mint Shoot Tips

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze‐avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet‐vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low‐temperature scanning electron microscopy, as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/?1°C than in plants cultivated at 25°C. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:707–717, 2013     

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein

Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20 degrees C x min(-1)). It also decreased with increasing seed crystal size under a slow cooling rate (0.01 degrees C x min(-1)), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4- and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25 degrees C below T(m), respectively. After annealing for 2 h at 0.25 degrees C below T(m), TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.     

Ultrastructural appearance of freeze-substituted schistosomula of Schistosoma mansoni frozen by a two-step cooling schedule

Schistosomula of the parasitic helminth Schistosoma mansoni were frozen by two-step cooling, then examined for ultrastructural changes by the freeze-substitution method. Samples were cooled at 1 °C min^?1 to ?20, ?25, ?28, and ?38 °C before being cooled at 10,000 °C min^?1 to ?196 °C. The results showed that progressive partial dehydration of the parasites occurred during slow cooling. Numerous cavities, indicating the presence of intracellular ice crystals, were observed in organisms which did not become shrunken. The sizes of the ice cavities varied between organisms and also within the same cell type in individual organisms indicating that intracellular ice nucleation may occur at any time during the slow cooling step. Some organisms cooled first to ?28 or ?38 °C contained no evidence of ice crystal formation. When correlated with previously reported infectivity studies, the results indicated that successful cryopreservation of schistosomula requires slow cooling to approximately ?30 °C to induce cryodehydration, followed by rapid cooling to ?196 °C to prevent ice nucleation or crystal growth.     

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.     

Studies on the optimal cooling rate for freezing human diploid fibroblasts

Ampoules containing each 1 ml of Dulbecco's Modified Eagle Medium with 16.6% fetal calf serum and 10% dimethylsulfoxide were insulated in various ways and placed into different cooling boxes. The resulting cooling velocities of the medium ranged from about 0.7 to 102 °C/min. As revealed by cellular attachment in recovery cultures, human diploid fibroblasts cooled at about 1.5 to 4.5 °C/min to − 78 °C prior to storage in liquid nitrogen showed an optimal survival of about 60%. Survival was about 25% at cooling rates of 0.7 and 19 °C/min, respectively. The optimal cooling rate was achieved by insulating the freezing ampoules with 1 to 3 closed vessels and placing them into a dry ice chest, or into a dry ice/ethanol bath.     

A differential scanning calorimeter for ice nucleation distribution studies--application to bacterial nucleators

A differential scanning calorimeter has been developed for the automatic detection and measurement of dropwise freezing within a sample of 100-200 water drops. A typical drop size of 1 microliter is employed. The sample is distributed on flat, square (4-cm) thermoelectric sensors and the temperature is scanned downward by conductive cooling to a liquid nitrogen bath. The rate of cooling, typically 1 degree C/min, is set by the choice of a heat conduction rod between the calorimeter and the liquid nitrogen bath. The voltages from the thermopiles along with a system temperature-measuring thermocouple are continuously monitored by digital voltmeters and recorded every half-second in a computer memory. A freezing event in a drop is detected by a characteristic voltage signal whose integral with time is proportional to the size of the drop and its heat of fusion. The half-life of a freezing event signal is 10 s for a 1-microliter drop. The integrated signal produced from multiple freezing events is shown to provide a direct measure of the number of drops frozen at a given temperature. A distribution curve and its smoothed derivative can be constructed directly from these measurements. The instrument, which is termed an "ice nucleometer," is illustrated in determining the ice nucleation distribution in a population of Escherichia coli harboring cloned ice nucleation genes.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

Freeze Concentration of Some Foodstuffs Using Ice Nucleation-active Bacterial Cells Entrapped in Calcium Alginate Gel

Cells of an ice nucleation-active strain of Ermnia ananas were entrapped in calcium alginate to prepare an ice-nucleating gel usable as ice nuclei for freeze concentration. The ice-nucleating gel was also adjusted as to specific gravity. When it was placed at a desired position in a liquid material such as egg white, ice formed at this position as the material was cooled. It was possible to put the ice- nucleating gel in liquid foodstuffs such as egg white and lemon juice before their temperatures reached subzero points. Application of this method produced freeze-concentrated foods whose properties were not significantly deteriorated.