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PubMed2006

Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties.

Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/ml. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.

Freeze Drying↗

PubMed2002

Effect of freeze-drying, freezing and frozen storage of blood plasma on fibrin network characteristics.

INTRODUCTION: We investigated the effect of freezing, freeze-drying and the duration of frozen storage of blood plasma on fibrin network characteristics of clots subsequently produced. MATERIALS AND METHODS: Fibrin network characteristics of clots made from freeze-dried and frozen plasma were compared to those made from fresh plasma. Freeze-dried pooled plasma was reconstituted and frozen each month for 4 months to describe the differences in fibrin networks that occur as a result of storage of the plasma over this period. RESULTS: Compared to freezing, freeze-drying of plasma had fewer undesirable effects on the fibrin network characteristics measured. Only the permeability of the clots from freeze-dried plasma was significantly less compared to the values of clots from the fresh plasma (p=0.005). Fibrinogen activity and mass-length ratio, compaction and fibrin content of the clots made from frozen plasma were, however, all significantly affected by freezing. Mass-length ratio and compaction showed a linear decrease and fibrin content a linear increase over a 4-month frozen storage period, thereby indicating that these variables were probably not stable. Large variation found in the data from each month indicates that there may be other factors, apart from storage time, that have a larger influence on these fibrin network characteristics, than frozen storage of plasma for 4 months. Storage of plasma in the freeze-dried form for 4 months resulted in a significant increase in fibrinogen (p=0.0004) but significant decrease in fibrin content (p=0.0002). CONCLUSIONS: Although the process of freeze-drying had fewer undesirable effects on the measured fibrin network characteristics compared to freezing, storage in both forms resulted in altered activity upon rehydration and thawing.

Blood Coagulation↗

PubMed2003

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

Animals↗

PubMed1984

On estimating freezing times during tissue rapid freezing.

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 micron depths in specimens. The calculations indicate that cooling rates in excess of 4 X 10(4) K s-1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 micron, the times obtained are 0.1-0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about +/- 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 microseconds, which can be taken as an upper limit for the resolution time for rapid freezing.

Freezing↗

PubMed2002

Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains.

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

Aquaporin 1↗

PubMed1994

Effects of temperature and freezing on hepatocytes isolated from a freeze-tolerant frog.

Metabolically active hepatocytes prepared from freeze-tolerant wood frogs, Rana sylvatica, were used to examine the direct effects of temperature and freezing on cryoprotectant synthesis and to assess the effectiveness of the natural cryoprotectant glucose in the freezing preservation of the isolated cells. Freshly isolated hepatocytes showed slow leakage of lactate dehydrogenase, readily synthesized urea, and oxidized a variety of 14C-labeled substrates. Effects of temperature on glucose production by isolated hepatocytes showed a normal Arrhenius relationship. However, compared with 0 degrees C control cells, either incubation at higher temperatures or freezing at -3 degrees C reduced the activity of glycogen phosphorylase alpha. These data suggest that the freezing-induced cryoprotectant production that occurs in vivo is not due to direct action of either low temperature or freezing on liver cell metabolism. The natural cryoprotectant glucose was also an excellent cryoprotectant of hepatocytes in vitro. In the absence of glucose, freezing caused a substantial leakage of lactate dehydrogenase from isolated hepatocytes, the rate of leakage increasing as freezing temperature decreased. Addition of 200-600 mM glucose to the incubation medium (similar to natural levels) fully protected cells against damage during freezing at -4 or -8 degrees C, normal freezing temperatures experienced by these frogs. Glucose also greatly improved freezing survival of isolated frog hepatocytes at ultralow temperatures (-80 or -196 degrees C).

Acclimatization↗

PubMed1992

Responses to freezing exposure of hatchling turtles Trachemys scripta elegans: factors influencing the development of freeze tolerance by reptiles.

Hatchling red-eared turtles Trachemys (= Pseudemys) scripta elegans (Wied) from a Louisiana population display a significant ability to withstand the freezing of extracellular body fluids. All animals survived at least 2 h of freezing at -2.5 or -4 degrees C. At -2.5 degrees C, survival declined to 50% after 6 h of freezing and no animals recovered after 24 h or longer, when mean ice content reached 54.7 +/- 1.4% of total body water. At -4 degrees C, all turtles recovered from 4 h of freezing exposure with a mean ice content of 49.6 +/- 2.4%, but survival dropped sharply thereafter with no animals recovering after 8 h, when ice content had reached 64.5 +/- 0.7%. Survival times were substantially shorter and percentage ice values greater than comparable values for hatchling painted turtles (Chrysemys picta (Schneider)) from northern populations subjected to identical freezing exposures. The ability to synthesize cryoprotectants in response to freezing was poorly developed in T. s. elegans; maximal accumulation of glucose was only 3.2 mumol g-1 wet mass in liver. Lactate content increased two- to threefold in oxygen-sensitive organs (heart and brain) during freezing, but levels of lactate and other putative cryoprotectants were unchanged in other organs. Total free amino acid content rose significantly in liver, muscle and blood during freezing; increased taurine concentration was primarily responsible for the changes in liver and blood. The capacity for freezing survival by T. s. elegans hatchlings from southern populations would be of limited use for hibernation in a cold climate, but the metabolic responses to freezing displayed by these animals might be enhanced by northern populations to increase their freeze tolerance.

Acclimatization↗

PubMed2006

Critical freezing rate in freeze drying nanocrystal dispersions.

Recent advances in nanoparticle technologies have significantly enhanced the oral and parenteral delivery of poorly water-soluble active pharmaceutical ingredients (APIs). However, reports have been limited on the various drying procedures to convert a liquid nanocrystal dispersions into solid dosage forms. The solid dosage form should consist of nanocrystals that can readily reconstitute into their original size upon dissolution in water. Herein, the freeze drying process of nanocrystal dispersions was examined at varying freezing rates (speed of freezing interface). As freezing rate decreases, more particle-particle aggregation developed. A critical freezing rate, below which the dried nanocrystals cannot be re-dispersed, was identified based on the plot of the particle size of reconstituted nanocrystals versus freezing rate. Freeze drying at a freezing rate near the critical value produces dry powders of bimodal particle size distribution after re-dispersion. In addition, API concentration was found to significantly affect the critical freezing rate and therefore the re-dispersibility of dry powders. The concept of critical freezing rate is critical for the development of solid dosage forms of liquid nanocrystal dispersions.

Crystallization↗

PubMed1997

Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure.

Some unicellular organisms present challenges to chemical fixations that lead to common, yet obvious, artifacts. These can be avoided in entirety by adapting spray-freezing technology to ultrarapidly freeze specimens for freeze substitution. To freeze specimens, concentrated suspensions of cells ranging in diameter from 0.5-30 pm were sprayed with an airbrush at 140-200 kPa (1.05-1.5 torr; 20.3-29.0 psi) into a nylon mesh transfer basket submerged in liquid propane. After freezing, the mesh basket containing the frozen sample was lifted out of the chamber, drained and transferred through several anhydrous acetone rinses at 188 K (-85 degrees C). Freeze substitution was conducted in 1% tannic acid/1% anhydrous glutaraldehyde in acetone at 188 K (-85 degrees C), followed by 1% OsO4/acetone at 277 K (4 degrees C). Freeze substitution was facilitated using a shaking table to provide gentle mixing of the substitution medium on dry ice. High quality freezing was observed in 70% of spray-frozen dinoflagellate cells and in 95% of spray-frozen cyanobacterial cells. These could be infiltrated and observed directly; however, overall ultrastructural appearance and membrane contrast were improved when the freeze-substituted cells were rehydrated and post-fixed in aqueous OSO4, then dehydrated and embedded in either Spurr's or Epon resin. Ultrastructural preservation using this ultrarapid freezing method provided specimens that were consistently superior to those obtainable in even the best comparable chemical fixations.

Animals↗

PubMed1999

The effects of freeze drying and freeze drying additives on the prothrombin time and the international sensitivity index.

AIM: To determine whether freezing, freeze drying protective additives, or freeze drying of plasma samples from patients on coumarin treatment and from normal individuals affects prothrombin times or the international sensitivity index (ISI) calibration. METHODS: The effect of the addition of the protective additives singly and combined on the prothrombin time of coumarin samples and normal samples before and after freeze drying was observed using high and low ISI reference thromboplastins. ISI values were also determined. RESULTS: Freezing caused a prolongation of prothrombin time in the normal plasma samples with both reagents, which was significant with the low ISI human. Prolongation (non-significant) of the prothrombin time in coumarin plasma samples occurred with the human reagent only. Significant prolongation of normal prothrombin time by some of the protective additives before and after freeze drying was observed with both thromboplastins but to a greater extent with the human. Significant prolongation of prothrombin time in coumarin plasma samples was observed, but again was more marked with human thromboplastin. An approximate ISI was determined on the 20 coumarin samples. The only marked ISI change was with the WHO human thromboplastin after freeze drying of plasma, where a decrease from 0.95 to 0.90 was observed, corresponding to a marked prothrombin ratio increase. CONCLUSIONS: Freeze drying additives and the freeze drying procedure prolong normal and coumarin prothrombin times, with low ISI thromboplastin. Less marked prolongations occurred with a high ISI rabbit reagent, coumarin samples showing more significant prolongations. Marked ISI change in freeze dried plasma was only recorded with the low ISI ECAA human reagent. Frozen normal plasma samples cannot be used with confidence for ISI calibrations.

Animals↗

PubMed2005

Role of glucose-6-phosphate dehydrogenase in freezing-induced freezing resistance of Populus suaveolens.

To explore the role of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the enhancement of freezing resistance induced by freezing acclimation, G6PDH was purified from the leaves of 8-week-old Populus suaveolens cuttings. The G6PDH activity in the absence or the presence of reduced dithiothreitol (DTT(red)) were determined, and the changes in superoxide dismutase (SOD), peroxides (POD) and cytosolic G6PDH activities, malondial-dehyde (MDA) content as well as freezing resistance (expressed as LT(50)) of P. suaveolens cuttings during freezing acclimation at -20 degrees C were investigated. The results showed that the purified G6PDH was probably located in the cytosol of P. suaveolens. Freezing acclimation increased the activities of SOD, POD and cytosolic G6PDH, and decreased the MDA content and LT(50) of cuttings, while 2 d of de-acclimation at 25 degrees C resulted in a decrease in SOD, POD and cytosolic G6PDH activities, and caused an increase in MDA content and LT(50). The change in cytosolic G6PDH activity was found to be closely correlated to the levels of SOD, POD and MDA, and to the degree of freezing resistance of cuttings during freezing acclimation. It is suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation is related to the distinct increase in cytosolic G6PDH activity, which may be involved in the activation of SOD and POD, and the induction of freezing resistance of cuttings.

Acclimatization↗

PubMed1996

Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure.

Using in parallel electron microscopy of ultrathin frozenhydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.

Crystallization↗

PubMed2006

Physical characterization of pharmaceutical formulations in frozen and freeze-dried solid states: techniques and applications in freeze-drying development.

Physical characterization of formulations in frozen and freeze-dried solid states provides indispensable information for rational development of freeze-dried pharmaceutical products. This article provides an overview of the physical characteristics of formulations in frozen and freeze-dried solid states, which are essential to both formulation and process development. Along with a brief description of techniques often used in physical characterization for freeze-drying development, applications of and recent improvements to these techniques are discussed. While most of these techniques are used conventionally in physical characterization of pharmaceuticals, some techniques were designed or modified specifically for studies in freeze-drying. These include freeze-drying microscopy, freeze-drying X-ray powder diffractometry and cryoenvironmental scanning microscopy, which can be used to characterize the physical properties of the formulation under conditions similar to the real vial lyophilization process. Novel applications of some conventional techniques, such as microcalorimetry and near infrared (NIR) spectroscopy, which facilitated freeze-drying development, receive special attention. Research and developmental needs in the area of physical characterization for freeze-drying are also addressed, particularly the need for a better understanding of the quantitative correlation between the molecular mobility and the storage stability (shelf life).

Animals↗

PubMed1976

A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation.

The ultrastructure of bull and boar spermatozoa was investigated following different cryopreparation methods and chemical fixation. Spray-freezing was used for cryofixation in both freeze-etching and freeze-drying studies. Freeze-etching of boar spermatozoa revealed that the arrangement of the postnuclear striations differed from that in the bull. Freeze-drying gave excellent results for structural preservation, which were equal to those of chemical fixation. Some structural details not visible in chemically fixed cells were detected in freeze-dried and vacuum-embedded bull and boar spermatozoa, e.g. the arrangement of the lamellar nuclear contents, known from freeze-fractures, and a fine lamellar structure of the acrosomal contents. Cryofixation by spray-freezing combined with freeze-drying makes any contact of the cells with fixatives, buffer solutions and dehydration media unnecessary, and potentially provides all the advantages of ultrathin sectioning required for histochemical studies.

Acrosome↗

PubMed2000

Hepatic changes in the freeze-tolerant turtle Chrysemys picta marginata in response to freezing and thawing.

Select hepatic changes in the freeze-tolerant hatchling turtle, Chrysemys picta marginata, were studied in response to freezing at -2.5 degrees C and thawing. Upon freezing, a small, selective increase in the liver weight with no increase in body weight was seen suggestive of an hepatic capacitance response. In all turtles studies, lobular differences in the hepatic content of glycogen were evident: the smaller lobe contained twice as much glycogen as the larger lobe. The response to freezing and thawing was comparable. Total hepatic glycogen levels of turtles were reduced approximately 60 per cent from control levels in the frozen state and recovered to >80 per cent of control levels in the thawed state. Compared to the control state, turtle blood glucose levels were: unchanged after 12 h in the cool state; reduced 28 per cent after 24 h and increased two-fold after 48 h in the frozen state; and increased 4.5-fold in the thawed state. Thus, changes in hepatic glycogen metabolism occur without large changes in blood glucose levels. In turtle liver plasma membranes, the hepatic alpha(1)-adrenergic receptor was barely detectable and did not change. The beta(2)-adrenergic receptor was expressed at high levels and, compared to control levels, was: unchanged after 12 h in the cool state; reduced 20 per cent after 24 h and 40 per cent after 48 h in the frozen state. On thawing, this receptor was 50 per cent of control levels. While catecholamines working through the beta(2)-adrenergic receptor may effect early hepatic glycogen breakdown in response to freezing, other factors must be involved to complete the process. The plasma membrane-bound enzyme gamma-glutamyltranspeptidase displayed a different pattern of changes indicative of selective modulation: it was increased 2.7-fold over control levels in the cool state; unchanged in the frozen state; and increased 1.8-fold in the thawed state. The activity of the kidney enzyme was decreased in the cool state and slightly increased in the frozen and thawed states emphasizing the tissue-specific nature of the changes in the activity of gamma-glutamyltranspeptidase in response to freezing and thawing. The similarities and differences of the hepatic changes in response to freezing and thawing in the freeze-tolerant hatchling turtle to those we have previously reported for the freeze-tolerant frog are discussed.

Animals↗

PubMed2002

Freeze-drying using vacuum-induced surface freezing.

A method of freezing during freeze-drying, which avoids undercooling of a solution and allows growth of large, dendritic ice crystals, was investigated. Aqueous solutions of mannitol, sucrose, or glycine were placed under a chamber vacuum of approximately 1 mbar at a shelf temperature of +10 degrees C. Under these conditions, the solutions exhibit surface freezing to form an ice layer of approximately 1-3 mm thickness. On releasing the vacuum and lowering the shelf temperature to below the freezing point of the ice in the solution, crystal growth occurs to yield large, chimney-like ice crystals. The duration of primary drying of a frozen cake--as measured by using inverse comparative pressure measurement--was up to 20% shorter than when using a "moderate" freezing procedure (2 K shelf temperature per min). With mannitol, however, the residual moisture content of the final dried product was higher than with moderate freezing, and with sucrose and glycine there was no difference. These findings are related to the structures of the dried cakes formed during freezing, as examined by light microscopy and wide-angle X-ray diffraction. The introduction of an annealing step (4 h at a shelf temperature slightly above the onset melting point of the ice in the frozen cake) combined with the vacuum-induced surface freezing procedure maintains the rapid primary drying and produces a low residual moisture (0.2%) for the freeze-dried mannitol solution.

Crystallization↗

PubMed1993

Cytoplasmic structure in organotypic cultures of rat hippocampus prepared by rapid freezing and freeze-substitution fixation.

We have compared rapid freezing followed by freeze-substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 microns without visible ice crystals. Freeze-substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze-substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques.

Animals↗

PubMed2003

Freezing curve-based monitoring to quickly evaluate the viability of biological materials subject to freezing or thermal injury.

This paper is aimed at investigating the roles of freezing dynamics of a liquid droplet to characterize the properties of the material. In particular, freezing curve-based monitoring was proposed to quickly evaluate the viability of biological materials subject to freezing, re-warming, or other kinds of injury, which is an extremely important issue in practices such as cryobiology, hyperthermia, or freshness evaluation of bio-samples. An integrated micro analysis device was fabricated which is simple in structure and cheap to make. Preliminary freezing results demonstrated that minor changes in a biological material due to freezing or warming injury might result in a significant deviation of its freezing curve from that of the intact biomaterials. Several potential thermal indexes to quantify the material features were pointed out. Further, experiments were performed on some freezing and thawing processes of small amount of water on a cooling surface to test the effects of droplet sizes, measurement sites, cooling strength, and cooling geometry, etc., on the freezing responses of a water droplet. Their implementation in developing a new micro analysis system were suggested. This freezing curve-based monitoring method may open a new strategy for the evaluation of biomaterials subject to destruction in diverse fields.

Animals↗