Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

Mechanism of cryoprotection by extracellular polymeric solutes.

To elucidate the means by which polymer solutions protect cells from freezing injury, we cooled human monocytes to -80 degrees C or below in the presence of various polymers. Differential scanning calorimetric studies showed that those polymers which protect cells best have a limiting glass transition temperature (T'g) of approximately -20 degrees C; those with a T'g significantly higher or lower did not protect. Freeze-etch electron micrographs indicated that intracellular ice crystals had formed during this freezing procedure, but remained smaller than approximately 300 nm in the same proportion of cells as survived rapid thawing. We propose that cryoprotection of slowly frozen monocytes by polymers is a consequence of a T'g of -20 degrees C in the extracellular solution. In our hypothesis, the initial concentration and viscosity of protective polymer solutions reduce the extent and rate of cell water loss to extracellular ice and limit the injurious osmotic stress, which cells face during freezing at moderate rates to -20 degrees C. Below -20 degrees C, glass formation prevents further osmotic stress by isolating cells from extracellular ice crystals, virtually eliminating cell water loss at lower temperatures. On the other hand, the protective polymer solutions will allow some diffusion of water away from cells at temperatures above T'g. If conditions are correct, cells will concentrate the cytoplasm sufficiently during the initial cooling to T'g to avoid lethal intracellular freezing between T'g and the intracellular Tg, which has been depressed to low temperatures by that concentration. Thus, when polymers are used as cryoprotective agents, cell survival is contingent upon maintenance of osmotic stress within narrow limits.     

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.     

Cryoprotection of spinach chloroplast membranes by dextrans

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity.Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions.From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.     

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms

Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.     

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.     

Interfacial free energies of intact and reconstituted erythrocyte surfaces: Implications for biological adhesion

Model cell surfaces consisting of phospholipids or phospholipids and the erythrocyte membrane glycoprotein glycophorin have been formed at an oil/water interface. Interfacial free energies have been estimated from surface wetting by both hydrophobic and hydrophilic test droplets on both the model surfaces and on intact erythrocytes. The use of a dense fluorocarbon oil to form the oil/water interface facilitates analysis by minimising surface deformation by the test drop. Hydrophobic test droplets (polar hydrocarbon oils) show increasing contact angles (decreasing wetting) with increasing hydrophilicity (decreasing interfacial free energy) of the model interface. Hydrophilic test droplets (phase separated aqueous polymer systems) show the opposite behaviour, spreading more as the interfacial free energy is decreased. Both systems give similar estimates of the interfacial free energy. Glycophorin reproduces the wetting properties of intact cell surfaces by reducing the lipid-water interfacial free energy from 5·10^?3 J·m^?2 to 1·10^?6 J·m^?2. From molecular considerations it is concluded that ‘cell surface free energy’ is an ambiguous term; its magnitude depends on the location of the interface in question. Thus, in a thermodynamic analysis of interactions at biosurfaces (such as cellular adhesion, chemotaxis or membrane fusion), the interfacial free energies may vary by more than three orders of magnitude depending on the location of the particular interface.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.

     

Long- and short-term freezing induce different types of injury in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaf cells

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in “fracture-jump lesions” in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.     

Alterations in membrane transport properties by freezing injury in herbaceous plants:

Leakage of ions from a thawed tissue is a common phenomenon of freezing injury. This leakage is usually assumed to be due to loss of membrane semipermeability or membrane rupture by freezing injury. Freeze injured, yet living, onion (Allium cepa L.) epidermal cells were used to study alterations in cell membranes that result in leakage of ions. In spite of a large efflux of ions, freeze injured cells could be plasmolysed and they remained plasmolysed for several days just like the unfrozen control cells. Injured cells also exhibited protoplasmic streaming. Passive transport of KCl, urea and methyl urea across the cell membranes of injured and control cells was also studied. No difference could be detected for the transport rates of urea and methyl urea between control and injured cells. However, a dramatic increase in the transport rate of KCl was found for the injured cells. Depending upon the extent of initial freezing injury, an increase or a decrease in injury symptoms was found in the post-thaw period. During the progress of freezing injury, 10 days after thawing, a swelling of the protoplasm was seen in the irreversibly injured cells. In spite of this swelling, these cells could be plasmolysed. It appears that the high amount of K⁺ that leaks out into the extracellular water, due to freezing injury, causes protoplasmic swelling by replacing Ca²⁺ in the plasma membrane. We conclude that protoplasmic swelling is a sign of secondary injury. The results presented in this study show that membrane semipermeability is not completely lost and membrane rupture does not occur during the initial stage of freezing injury. In fact, the cells have the ability to repair damage depending upon the degree of injury. Our results show there are specific alterations in membrane semipermeability (e.g., transport of K⁺) which could be repaired completely depending on the degree of injury. These findings suggest that ion leakage due to freezing injury is due to alteration in the membrane proteins and not in the membrane lipids.     

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.     

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.     

Transport phenomena during freezing of adipose tissue derived adult stem cells

In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the "best-fit" membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.     

Nucleation and growth of ice in deeply undercooled erythrocytes

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.     

Cryoprotective effect of polyols on rat embryos during two-step freezing.

The cryoprotective effect of polyols on rat embryos was measured after two-step freezing, and the mechanism of action of polyols on embryo survival was examined. Rat embryos frozen in solution of polyol by two-step method at the morula stage showed higher survival than that obtained using DMSO. As the number of hydroxyl groups increased, the cryoprotective effect of the polyol increased. However, this was true only when the additive could permeate the cell membrane. Of the additives tested, four or five carbon polyols were most effective at concentrations of 0.3 or 1.0 M than two, three, six, or seven carbon polyols. The highest survival rate was obtained with adonitol, which yielded 83% embryo survival at 1.0 M and 67% even at 0.3 M. Embryos frozen in 0.3 M adonitol and transferred directly into foster mothers without any dilution of the additive after thawing developed into live young. During slow cooling below -40 degrees C, embryonic blastomeres exhibited cell fusion only in the presence of adonitol. These findings suggest that one cryoprotective action of polyols is that the hydroxyl groups act both on the cell surface and the cytoplasm to stabilize the bound water on the embryonic membrane, and that the length of the C-chain determines the permeability of the membrane to the additive.     

Cryopreservation of platelets using trehalose: The role of membrane phase behavior during freezing

In blood banks, platelets are stored at 20–24°C, which limits the maximum time they can be stored. Platelets are chilling sensitive, and they activate when stored at temperatures below 20°C. Cryopreservation could serve as an alternative method for long term storage of platelet concentrates. Recovery rates using dimethyl sulfoxide (DMSO) as cryoprotective agent, however, are low, and removal of DMSO is required before transfusion. In this study, we have explored the use of trehalose for cryopreservation of human platelets while using different cooling rates. Recovery of membrane intact cells and the percentage of nonactivated platelets were used as a measure for survival. In all cases, survival was optimal at intermediate cooling rates of 20°C min^?1. Cryopreservation using DMSO resulted in high percentages of activated platelets; namely 54% of the recovered 94%. When using trehalose, 98% of the platelets had intact membranes after freezing and thawing, whereas 76% were not activated. Using Fourier transform infrared spectroscopy, subzero membrane phase behavior of platelets has been studied in the presence of trehalose and DMSO. Furthermore, membrane hydraulic permeability parameters were derived from these data to predict the cell volume response during cooling. Both trehalose and DMSO decrease the activation energy for subzero water transport across cellular membranes. Platelets display a distinct lyotropic membrane phase transition during freezing, irrespective of the presence of cryoprotective agents. We suggest that concomitant uptake of trehalose during freezing could explain the increased survival of platelets cryopreserved with trehalose. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Cryopreservation of bovine somatic cells using antifreeze polyamino-acid (carboxylated poly-l-lysine)

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, obtained by converting 65 mol% of amino groups to carboxyl groups after synthesizing ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective properties similar to those of anti-freeze protein. The addition of CPLL to freezing medium has been reported to improve the post-thawing survival rate of murine cells, human induced pluripotent stem (iPS) cells, embryonic stem (ES) cells and embryos. In this study, investigating CPLL for its effectiveness as a new cryoprotective material is aimed. In experiments with bovine somatic cells, CPLL was suggested to have an equal or superior cryoprotective effect to dimethyl sulfoxide (DMSO), the conventional material for cellular frozen storage, based on the results for post-thawing cell survival and proliferation rates. CPLL was demonstrated to have another advantage; thawed cells can be cultured without removing the cryopreservation medium when CPLL is used, but not when DMSO is used. These results suggest that CPLL could be used as cryoprotective material for bovine cells. It is also expected that CPLL can be applied to embryo and oocytes storage for cattle, and similar functions for cells and embryos of other animal species.     

Post-thaw suspension of red cells cryopreserved with hydroxyethyl starch.

Post-thaw suspension of red cells cryopreserved with hydroxyethyl starch (HES) gives rise to an increase in the quantity of supernatant hemoglobin and a reduction in cell recovery. This occurs regardless of the suspension media used but may be delayed by some solutions. Extended suspension with most solutions results in loss of intracellular contents from a number of cells. The resulting ghosts account for 12–14% of the total cell population. In the presence of 14% HES, deposits on the surface of damaged cells indicate that cellular contents are adherent to the cell membrane and therefore do not contribute to the free hemoglobin in the external solution. The result is a misleading high cell recovery value and suggests that this test does not accurately describe true cell damage in the presence of HES. Since the saline stability of suspended cells changes very little from that prior to suspension (at thaw), its value is a more accurate assessment of cell damage.     

Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF₁) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.     

Freezing tolerance of sea urchin embryo pigment cells

Various stresses, including exposure to cold or heat, can result in a sharp increase in pigmentation of sea urchin embryos and larvae. The differentiation of pigment cells is accompanied by active expression of genes involved in the biosynthesis of naphthoquinone pigments and appears to be a part of the defense system protecting sea urchins against harmful factors. To clarify numerous issues occurring at various time points after the cold injury, we studied the effect of shikimic acid, a precursor of naphthoquinone pigments, on cell viability and expression of some pigment genes such as the pks and sult before and after freezing the cultures of sea urchin embryo cells. The maximum level of the pks gene expression after a freezing–thawing cycle was found when sea urchin cells were frozen in the presence of trehalose alone. Despite naphthoquinone pigments have been reported to possess antioxidant and cryoprotectant properties, our data suggest that shikimic acid does not have any additional cryoprotective effect on freezing tolerance of sea urchin embryo pigment cells.