Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.     

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

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

Three-step cooling: A preservation method for adult rat heart cells

Adult rat heart cells were exposed to two-step cooling to ?196 °C with different holding periods at different subzero temperatures between both steps. The highest survival based on the percentage of trypan blue-excluding cells was 25% with 10% DMSO and a holding period of 6 min, and 21% with 15% DMSO and a holding period of 30 min. The highest survival based on morphological intactness was about 10%; there was no difference in results after cooling with 10 and 15% DMSO, and after holding between 2 and 30 min. The optimal survival based on the percentage of contracting cells was 52%, with 15% DMSO and a holding period of 2 min.When the holding period was replaced by a programmed cooling stage, the results could be improved. With this threestep cooling method, the optimal values, based on the number of trypan blue-excluding, intact, and contracting cells, were 40, 32, and 60%, respectively. It appeared that in the presence of 10% DMSO, which provided better survival than 5 and 15%, no significantly different results were obtained when the starting temperatures of the second cooling step varied between ?10 and ?20 °C, when the end temperatures varied between ?30 and ?60 °C, or when the cooling rates of the second cooling step varied between 0.1 and 1 °C/min. Three-step cooling provided similar results as linear cooling from 0 to ?100 °C, followed by rapid cooling to ?196 °C.     

Regeneration of Plants from Cell Suspensions Frozen at −20, −70 and −196°C

Cell suspensions of carrot, Datura, tobacco and soybean subjected to ?20°C, ?70°C and ?196°C in the presence of a suitable cryoprotective agent, and stored for various lengths of time have been revived. After revival these cells divided to form callus masses. Direct immersion in liquid nitrogen invariably killed the cells, whereas cooling at the rate of 1 or 2°C/min, or pre-freezing briefly at ?20 and ?70°C, followed by freezing at ?196°C retained the viability. Depending on the plant species up to 70% of the cell clumps could withstand ultra-cooling. Tobacco and Datura cell suspensions were more sensitive to cold treatment than were those of carrot. Actively growing cell suspensions containing small cell-clumps revived rapidly, while filtered cell-suspensions of free cells only occasionally survived. Calli of tobacco and carrot obtained from frozen suspensions have been regenerated into plants.     

Pancreatic islet banking: The transplantation of frozen-thawed rat islets transported between centers

This report describes the feasibility of islet banking for the purpose of transporting isolated islets from one center to another for transplantation. Adult rat islets survived freezing to ?196 °C when 0.25 °C/min was used as the cooling rate, 7.5 °C/min as the warming rate, and when the hyperosmotic protective agent was carefully removed. We were able to show that islets isolated and frozen in one center and transplanted in another center returned diabetic animals to clinical normalcy (fasting normoglycemia, aglycosuria, and weight maintenance). However, as measured by a glucose tolerance test three months after transplantation, these animals had an impaired early insulin release when compared with animals who received fresh islet transplants. Diabetic animals that received islets frozen at a cooling rate of 1.0 °C/min remained diabetic as measured by our clinical parameters. Thus specific definition of conditions used for cryopreservation is important in developing methods suitable for islet banking.     

Comparisons of glucose,sucrose, and dimethyl sulfoxide as cryoprotective agents for Babesia rodhaini, with estimates of survival rates

Comparisons were made between glucose, sucrose, and dimethyl sulfoxide (DMSO) as cryoprotective agents for the hemoprotozoan parasite, Babesia rodhaini, using infectivity for mice as the criterion of survival. Concentrations of the cryoprotectants tested were from 0.1 to 0.5 M for the sugars, and 1.5 to 2.5 M for DMSO. Glucose and sucrose were comparable as cryoprotectants, although glucose reduced infectivity of the parasites slightly more than did sucrose at above-freezing temperatures. When sucrose and DMSO were compared for cryoprotection during cooling to ?196 °C at nominal rates of 5, 100, and 500 °C/min, parasite survival varied with the type and concentration of cryoprotectant, but was higher in blood containing DMSO at all three cooling rates. The percentages of parasites that survived cooling at 100 °C/min and frozen storage in the presence of DMSO ranged from 20 to 36%.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. II. Warming rate and concentration of dimethyl sulphoxide.

Chinese hamster tissue culture cells in dimethyl sulphoxide (5%) required a lower holding temperature (?35 °C) for optimal survival on slow warming from ?196 °C using a two-step cooling schedule, compared with that required (?25 °C) when warming was rapid. A lower concentration of dimethyl sulphoxide (1%) did not affect the “protection” against damage on cooling from the holding temperature to ?196 °C and thawing. The results suggest that protective agents allow cells to be cooled initially to the holding temperature and minimize damage at the holding temperature. Damage following subsequent cooling and thawing may thus occur mainly as dilution shock on rewarming. This can be minimized by allowing the cells to shrink at the holding temperature.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

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.     

Survival of frozen-thawed human red cells as a function of cooling and warming velocities

Human red cells were equilibrated for 30 min at 20 °C in buffered saline containing 2 m glycerol and then frozen to ?196 °C at 0.27, 1.7, 59, 180, 480, 600, and 1300 °C/ min and warmed at 0.47, 1, 26, 160, and 550 °C/min. Cells frozen at 600 and 1300 °C/min responded in the classical fashion for cells containing intracellular ice; i.e., survivals were low when warming was slow (<10%), but increased progressively with increasing warming rate. The sensitivity to slow warming presumably reflects the recrystallization of intracellular ice. Cells frozen at 59 and 180 °C/ min yielded high survivals at all warming rates. This response is also consistent with the findings for other cells cooled just slowly enough to preclude intracellular ice. Cells frozen very slowly at 0.27 and 1.7 °C/ min, however, responded differently; survivals were considerably higher when warming was slow (0.47 or 1 °C/min) than when it was 26, 160, or 550 °C/min. This response is analogous to that observed recently by others in mouse embryos and in higher plant tissue-culture cells and to that observed for many years in higher plants. It also confirms previous observations of Meryman in human red cells. It may reflect osmotic shock from rapid dilution but, if so, the basis of the osmotic shock is uncertain.     

Interaction of rate of latent heat removal during freezing and rates of subsequent cooling and warming on survival of the blood protozoan, Babesia rodhaini

Babesia rodhaini parasites in murine blood containing 1.5 m DMSO were frozen at two rates, as judged by the duration of the “freezing plateau”, then cooled to ?196 °C and rewarmed at two rates to detect interactions between the duration of the plateau and rates of subsequent cooling and rewarming. Infectivity tests showed that fast and slow freezing (plateau times of about 1 sec and 30 sec, respectively) had similar effects on parasite survival when cooling was at 130 °C/min and warming was at 800 °C/min. However, when either the cooling rate was increased to 3500 °C/min or the warming rate was decreased to 2.3 °C/min, fast freezing decreased parasite survival more than did slow freezing. It is suggested that fast freezing accentuated the damaging effects of fast cooling and slow warming by increasing intracellular ice formation.     

Cryopreservation of monogonont rotifers

Development of techniques to maintain viable rotifer clones in a frozen state would preserve the genotype and reduce routine maintenance for those clones not being actively studied. To this end we have frozen Brachionus plicatilis in dimethyl sulfoxide at concentrations ranging from 6% to 18%. Survival rates decreased as the endpoint temperature was reduced from ?20 °C to ?45 °C, but did not decrease when the temperature was further reduced to ?196 °C (liquid nitrogen). Only 2% of the individuals survived freezing in liquid nitrogen.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to -196 degrees C

Survival of the parasitic protozoan, Babesia bigemina, in blood cooled at widely different rates to ? 196°C. International Journal for Parasitology4: 169–172. The infectivity of Babesia bigemina in blood containing 2 m DMSO was tested in 99 cattle after the blood had been cooled to ? 196°C at eight rates ranging from 0·73–3070°C/min. Blood cooled at each rate was infective; 95 of the recipients became infected, the exceptions being four of the seven cattle inoculated with blood cooled at 3070° C/min. The infectivity of blood cooled at 39, 82 and 212°C/min was higher than that of blood cooled at slower or faster rates. Least depression of infectivity occurred at 82°C/min.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

Liquid nitrogen vapour freezing of mouse embryos

Mouse morulae were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7 degrees C after seeding into liquid nitrogen vapour at -170 to -180 degrees C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M-glycerol, 2 M-propylene glycol, 2 M-ethylene glycol; 5-30 min equilibration time at 0 degrees C; 3-60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.     

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at ?196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10⁹ granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at ?196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at ?196 °C with high cellular recovery and at least 50% phagocytic function.     

Preservation of mouse embryos by two-step freezing

Eight-cell mouse embryos in 1.5 M DMSO were preserved in LN₂ by a two-step procedure. Fifteen minutes exposure at ?20 °C protected the embryos against damage during rapid cooling to -196 °C and during rapid warming and rapid dilution. Since survival was poor on slow warming it is suggested that the method permits the formation of some intracellular ice.