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.     

Optimal temperature ranges for control of cooling rate.

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between ?5 and ?20 °C followed by rapid cooling to ?196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to ?30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at ?196 °C.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

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.     

The effect of freeze rate,duration of phase transition,and warming rate on survival of frozen canine kidneys

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2–4 °C/min) to ?22 °C and thawed (70–110 °C/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25–1.0 °C/min) and either rapidly or slowly warmed (20–30 °C/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy.Cooling rates of 0.1 and 10 °C/min were too harmful to the kidney to have renal function after reimplantation.The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2–4 °C/min cooling rate following rapid warming (70 °–110 °C/min).Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r = 0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting postthaw survival of canine kidneys.     

The development of the cell cryopreservation protocol with controlled rate thawing

Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing–thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

Viability of frozen rat granulocytes and granulocyte precursors

Mature rat polymorphonuclear leukocytes (PMNs) were frozen to ?196 °C, thawed, and tested for functional viability using a variety of criteria. The assays for functional viability included: qualitative and quantitative nitroblue tetrazolium tests for phagocytic activity, fluorometric tests for membrane integrity, chemotaxis, and bactericidal activity. Maximal survival was obtained when mature PMNs were frozen in the presence of 10% dimethyl sulfoxide (Me₂SO) and 5% hydroxyethyl-starch (HES) for cells cooled at ~10 °C per minute, followed by rapid warming. Maximal survival was obtained for granulocyte precursor cells (as measured by CFU-c) after freezing in the presence of 10% Me₂SO and cooling at ~10 °C per minute. The principal new findings for mature PMNs were: (i) there was a synergistic effect between intra- and extracellular protective additives; (ii) the optimal cooling rate increases from approximately 0.3 to 10 °C per minute when an extracellular protective agent, such as HES is included in the freezing media; (iii) the zwitterion buffer Hepes has a small but consistently beneficial effect on survival; (iv) granulocytes obtained from peripheral blood consistently show a higher functional survival after freezing (95%) than do PMNs obtained from a glycogen-induced peritoneal exudate (70%); (v) neither serum, plasma, nor other macromolecules are needed in the postt-haw dilution media to obtain high survival; and (vi) cells frozen using an optimized two-step protocol survived as well as those frozen using a continuous cooling protocol.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.     

Parameters of biological freezing damage in simple solutions: Catalase: II. Demonstration of an optimum-recovery cooling-rate curve in a membraneless system

Seeded solutions of catalase in neutral 10 mM potassium phosphate buffer exhibited characteristic rate dependencies for freeze-thaw damage: Damage increased as the cooling rate was increased, and as the warming rate was decreased. The pattern of warming-rate dependence was independent of the prior cooling rate and also of the addition of KCl or of NaCl to the buffer. In contrast, the cooling-rate curve became almost flat upon addition of 0.1 M KCl, suggesting increased damage from concentrating solute at low cooling rates. In the presence of added NaCl, frank optimum-recovery cooling-rate curves were generated. At low NaCl levels (less than 10 mM) the optimum occurred at 0.5 °C/ min; at 27 and 81 mM NaCl, the optimum shifted to 5 and 20 °C/min, respectively. By comparison with KCl, it appears that the major factor causing damage at low cooling rates in NaCl is acidification. The factor causing damage at high cooling rates remains obscure. The argument that it is due to the trapping of the enzyme molecules at interfaces at high dilution, to be subsequently damaged by shearing stress or dehydration during the recrystallization attending slow warming, is mitigated by the finding that inactivation remains a function of the initial enzyme concentration at all cooling rates. The possibility that a particular conformational state is trapped in an unfavorable temperature zone was also considered: Three simple models were formulated, and the relative order of recovery was deduced for the possible sequences of fast and slow cooling and warming. The permutation observed for catalase was inconsistent with any of these three mechanisms, although they may be pertinent for the red cell and other systems. A final possibility, not yet explored, is that rapid cooling causes damage by producing nonequilibrium freezing, with large deviations of pH and/or solute concentration from those expected at equilibrium.     

Changes in cell size during the cooling, warming and post-thawing periods of the freeze-thaw cycle.

Chinese Hamster Ovary (CHO) cells were cooled at 1 and 200 °C/min and subsequently thawed, while being studied with a cryomicroscope. Post-thaw size changes were measured with a Quantimet 720 Image Analysing Computer. It was found that the behavior of individuals in a population varied and depended on cooling rate. Cooling at 1 °C/min resulted in cells showing no intracellular ice, whereas cooling at 200 °C/min caused intracellular ice formation in some cells but not in others. In addition, at the slow rate, during cooling, the cells shrank significantly but swelled on thawing to become larger than non-frozen controls. Following swelling, as their temperature rose, the cells shrank to the size of non-frozen controls. At the fast rate, cells showed variation in their amount of intracellular ice and in their degree of shrinkage. Cells containing most ice shrank least. On warming, cells with intracellular ice began to swell at a lower temperature than did those cells without intracellular ice, while after thawing they swelled to a greater extent partly due to widespread blebbing. Corresponding recovery indices were measured, and correlation of these with the above effects suggests that: (i) cells completely filled with intracellular ice are non-viable; (ii) cells partially filled with intracellular ice respond to, or can be rescued by, first warming; (iii) cells without intracellular ice are viable; (iv) viable cells are those which regain their original size following thawing; (v) non-viable cells are those which remain swollen above their original size.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol

1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.     

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.     

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.     

Cryogenic preservation of isolated islets of Langerhans: Two-step cooling

Rat islets of Langerhans were frozen to ?196 °C using a two-step freezing procedure. Islets isolated from the pancreases of Long Evans hooded rats were exposed to CMRL 1066 media containing 1 M dimethyl sulfoxide for 6 min at 4 °C. They were transferred directly to subzero holding baths ranging from ?20 to ?43 °C for 5 to 20 min prior to transfer to and storage in liquid nitrogen. After warming at ～7 °C/min, the islets were diluted with Hanks' balanced salt solution containing 10% fetal calf serum, washed, and cultured overnight. In general, maximum protection of the islets from the stress of cooling to ?196 °C was obtained after holding the islets at ?35 or ?40 °C for between 5 and 15 min. After thawing, islets frozen using an “optimized” two-step protocol released insulin in response to a glucose challenge at a rate equivalent to that of control islets.     

Cryopreservation of cell line Paesun by a rapid cooling

The purpose of the present study was to clarify the possibility of a rapid cryopreservation for cell line Paesun by cooling in the range of 30–40 °C/min to vapor phase of −120 ∼-140 °C before immersion into liquid phase of liquid nitrogen using 10% Me₂SO. After thawing, these cells were examined with assaying viability by trypan blue exclusion staining and survival by cloning in monolayer; the percentages of cell and colony recovery obtained in rapid cooling had a tendency to be lower than that by slow cooling of 1 °C/min but there were no significant differences between them. In addition, post-thaw cells were examined by assaying proliferation and susceptibility to virus lines; there were no significant differences between before and after cryopreservation. In conclusion, these findings indicate that Paesun can be successfully cryopreserved by the rapid cooling rate of 30 °C–40 °C/min.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Freezing tolerance in relation to cooling rate in an adult insect

In the adult tenebrionid beetle Upis ceramboides unusually low cooling rates are required to demonstrate maximum freezing tolerance, and a very slight change in rate can reduce survival from 100 to 0%. Freezing to ?50 °C results in 100% mortality at rates above 0.35 °C/min, but no injury is apparent if the cooling rate is 0.28 °C/min. The lower lethal temperature, determined with a cooling rate of 0.17 °C/min, is about ?60 °C. The maximum cooling rate which allows full survival is nearly identical to optimal cooling rates previously found for mouse embryos and some lymphocytes, but the striking sensitivity to very slight changes in rate is unique to Upis. Most studies dealing with insect freezing tolerance have utilized rates of 1 °C/min or faster, and the failure of some of these laboratory studies to observe freezing survival may be due to the use of lethal cooling rates.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.