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.     

Viability of spores after repeate freezing and thawing shocks

Survival of spores of the fungus Rhizopus nigricans after repeated freezing and thawing was investigated. The cooling rate was 10(4) degrees C/min. Dry spores were fully inactive after 32 repeated shocks. About one-half of spores were killed after 8 repetitions. The water content did not change the resistance, swollen spores reacted to shocks much like dry ones. The sensitivity of spores to freezing-thawing shocks increased considerably when the spores changed from the dormant to the active state. Already after a 30 min cultivation of spores in the nutrient medium two freezing and thawings were sufficient for inactivation of 60% spores. After a 90 min cultivation one freezing and one thawing were sufficient to inactivate practically all spores.     

Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.     

Recovery after freezing and thawing of cultured human diploid fibroblasts

Fibroblast strains established from donors differing in age, sex, and genetic disease were frozen and thawed under variable conditions and cell survival was determined. The cell density of the monolayer prior to freezing was found to be the most important parameter for optimal cell recovery after freezing to ?196 °C and thawing. We obtained the best results with exponentially growing cells at about half the individual saturation density. Cell recovery was influenced neither by parameters defined by the donor of the skin biopsy, nor by the number of passages during the exponential growth phase, nor by repeated trypsinization and freezing. Application of different linear cooling velocities which were attained by a novel programmable freezing system yielded similar cell survival rates within a wide range from 0.05 to 10 °C/min.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Ultrastructure of spores ofRhizopus nigricans after repeated freezing and thawing shocks

Disintegration of nuclear envelopes is the only ultrastructural change detectable by freezeetching in dormant spores of the mouldRhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.     

The prevention of erythrocyte swelling upon dilution after freezing and thawing

Cellular swelling of erythrocytes exposed to Me2SO during freezing and thawing may lead to hemolysis upon dilution of the cryoprotectant with pure electrolyte buffer. Excessive cell swelling is effectively avoided by exposing the RBC to the nonpenetrating sorbitol after thawing and before dilution. Due to the initial reduction in volume by sorbitol, cell swelling upon dilution may not cause hemolysis particularly with concentrations of 0.05 to 0.15 M of sorbitol in the diluting electrolyte buffer. Membrane damage incurred during freezing and thawing is particularly pronounced with the older red cell population, while the younger population membrane integrity can be preserved to an optimal degree.     

Elevated frequencies of micronuclei in cultured fibroblasts after freezing and thawing

Spontaneous micronuclei (MN) were determined in 69 fibroblast lines in the first subculture after cryoconservation. 45 cultures (65%) showed micronuclei in the normal range (less than 10 MN/500 cells), but 24 (35%) exhibited an elevation up to 60 MN/500 cells. In order to determine whether freezing and thawing is responsible for the enhanced MN level we studied the persistence of elevated MN in 10 cell cultures after freezing and thawing, and found that the MN levels returned to normal after 3 subcultures. In addition, the micronucleus formation before and after freezing was investigated in 2 newly established cell cultures obtained from probands whose cells had a particularly high number of MN. Both cultures had regular MN levels before freezing but a more than doubled number of MN when frozen samples were recultivated. This effect of the freezing procedure was confirmed with 5 separate biopsies obtained from the same donor. Since a freezing-related increase in MN was constantly observed in cells from some individuals but not in others, it might be possible that it is a constitutive trait which causes cultured fibroblasts of some individuals to be hypersensitive to the freezing and thawing procedure. It is also noteworthy that fibroblasts from 8 out of 12 probands with a familial malignant melanoma exhibited this phenomenon.     

Stability of granulocytosis-promoting activity of post-pheresis plasma on freezing and thawing.

Pretreatment of rats with fresh post-pheresis plasma (PPP) obtained by filtration leukopheresis results in a significant increase in the yield of granulocytes. The practical application of this method in human beings requires a period of storage of the PPP without substantial loss of its activity. These studies demonstrate the maintenance of granulocytosis-promoting activity of PPP for at least 2 weeks after storage at ?150 °C.     

Permeability changes in membranes of Neurospora crassa after freezing and thawing.

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [¹⁴C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.