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.     

Escherichia coli viability in an isochoric system at subfreezing temperatures

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between −5 °C and −20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to −10 °C and −15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at −20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at −15 °C and −20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.     

Freezing behavior of free protoplasts of winter rye

Free protoplasts prepared from the epicotyls of nonhardened rye seedlings were subjected to fast and slow freezing on a microscope-adapted thermoelectric stage. During rapid freezing to ?12 °C, ice formation occurred inside the protoplasts causing lethal disruption of cell and membrane organization. Under slow freezing to ?12 °C, ice formation occurred outside the protoplast with accompanying dehydration and contraction of the protoplast. Complete rehydration and recovery of the protoplasts occurred upon thawing after slow freezing. Free protoplasts therefore afford a new system for the study of mechanisms of plant cell freezing injury and resistance free of the complications presented by a cell wall.     

The contribution of the cryoprotectant to total injury in rabbit hearts frozen with ethylene glycol.

Following the failure of hearts to recover function after freezing at ?20 ° in the presence of 3 m ethylene glycol, a variety of experimental treatments was devised to determine the relative harmfulness of ice, high concentrations of electrolytes and high ethylene glycol concentration. Neither cooling to ?20 °C without freezing in a Ca²⁺-free solution containing twice the normal salt concentration and 6 m ethylene glycol (freezing 3 m ethylene glycol at ?20 °C doubles the solute concentration in the liquid phase), nor perfusion at ?1 °C with this solution were conducive to the recovery of hearts. However, perfusion with Ca²⁺-free 3 m ethylene glycol solution with twice the normal concentration of salts did allow full recovery of function, whereas perfusion with Ca²⁺-free 6 m ethylene glycol solution with normal salt concentrations did not. Therefore, the high ethylene glycol concentration encountered during freezing was the main cause of damage.     

Survival of frozen-thawed human red cells as a function of the permeation of glycerol and sucrose

Previous studies have demonstrated that glycerol does not have to permeate bovine red cells to protect them against subsequent freezing and thawing. The present study is concerned with the relation between solute permeation and freezing injury of human red cells. Cells were held in 2 m glycerol for 30 sec to 10 min at 0 °C and then frozen to ?196 °C at 60 °C/min. Cells cooled at this rate have a very low probability of undergoing intracellular freezing. Percent survivals (≡percent unhemolyzed) increased by 21% (from 66 to 80%) over the first 3-min period. Extrapolation to zero time (and zero glycerol permeation) yields a survival of 57%. Between 30 sec and 3 min the calculated osmolal ratio of intracellular glycerol to other solutes increased 240% (from 2.5 to 5.7). The human red cell is impermeable to sucrose at 0 °C. Cells suspended in 1.40 m sucrose (equiosmolal to 2.0 m glycerol) for 0.5 to 10 min prior to freezing yielded as high survivals after thawing as did cells in glycerol.These data indicate that prior permeation of additive is not a prerequisite for the survival of red cells subjected to subsequent freezing and thawing. Although sucrose and glycerol protect equally well to this point, differences appear when attempts are made to remove the additive. Over 90% of the cells survive the removal of glycerol. Only some 30% survive the removal of sucrose. Cells frozen in an equisomolal solution of sodium chloride do not even survive the initial freezing and thawing.The findings indicate that slow freezing injury cannot be accounted for in terms of the attainment of a critical minimum volume, nor can it be considered to be equivalent to posthypertonic hemolysis.     

Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aeroterrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic — South Shetlands, King George Island, Arctic — Ellesmere Island, Svalbard, Central Europe — Slovakia) were studied. Each strain was exposed to several freezing (−4°C, −40°C, −196°C) and desiccation (+4°C and + 20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to −196°C was the most harmful regime for all studied strains. Freezing at −4°C did not influence the survival of studied strains. Further, freezing down to −40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at −40°C, as well as desiccation at +20°C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to −40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia. This paper is dedicated to the memory of the late Dr. Bohuslav Fott (1908–1976), Professor of Botany at the Charles University in Prague, to mark the centenary of his birth.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.     

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.     

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.     

Biological activity of Friend spleen focus-forming virus after cold storage

Two stocks of Friend spleen focus-forming virus (SFFV) were prepared, one in saline and the other in Eagle's medium with 2% fetal calf serum, and the effects of different freezing, storage and thawing temperatures were determined for the recovery of infectious virus from each diluent. Once frozen, virus maintained its titer at ?70 and at ?170 °C for up to 13 weeks, while it lost titer at ?13 °C more rapidly if it had been prepared in saline than in medium. However, during the freezing process lower ambient temperatures (?70 and ?170 °C) gave lower virus yields than a higher temperature (?13 °C) did. Similarly, rapid thawing (in a 37 °C water bath) was less efficient than slow thawing (in 4 or 20 °C air) for the recovery of infectious SFFV, This study illustrates the importance, for efficient recovery of leukemogenic activity from stored murine leukemia virus stocks, of the temperature used for freezing or thawing, as well as for storage.     

Cold tolerance of a New Zealand alpine cockroach, Celatoblatta quinquemaculata (Dictyoptera, Blattidae)

Abstract. Ecophysiological features, including survival and recovery from freezing and determination of the freezable water content, are reported for a cold-adapted cockroach Celatoblatta quinquemaculata Johns 1966 (Dictyoptera, Blattidae) inhabiting alpine communities at altitudes greater than 1300 m a.s.l. in mountains of Central Otago, New Zealand. Nymphs ranged from 15 to 51 mg live weight of which 67% was water. Cockroaches had a mean supercooling point temperature of ?5.4 ± 0.1°C; with recovery from freezing close to this temperature being rapid, but no recovery was observed when frozen at ?9 to ?10°C. The duration of exposure to freezing conditions and the time allowed for recovery (24–96 h) both influenced individual recovery and subsequent survival. Comparison of supercooling point data and survival shows that this species possesses a few degrees of freeze tolerance, and individuals have been found frozen in the field when subzero temperatures occur. Differential scanning calorimetry showed ≈ 74% of body water froze during cooling and between 24 and 27% of total body water was osmotically inactive (unfreezable under the experimental conditions). Carbohydrates, other than glucose at 7.5μg/mg fresh weight, were in low concentrations in the body fluids, suggesting little cryoprotection. No thermal hysteresis from antifreeze protein activity was detected in haemolymph samples using calorimetric techniques. It is suggested that slow environmental cooling rates, together with high individual supercooling points, confer a small amount of freezing tolerance on this species enabling it to survive low winter temperatures. This has allowed it to colonize and maintain populations in alpine habitats > 1300 m a.s.1. in New Zealand.     

Recovery of a marine dinoflagellate following controlled and uncontrolled freezing

A free-living, marine dinoflagellate, Crypthecodinium cohnii, was successfully preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a 1 °C/min cooling rate. After storage for a minimum of 7 days at ?150 °C material frozen by this method demonstrated a 47.7% mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at ?55 °C for 1 hr. This material demonstrated a mean recovery of 30.8% with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after uncontrolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 68% recovery following controlled freezing.The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cause.     

Cryopreservation of erythropoietin-responsive cells in murine hematopoietic tissue

The optimal conditions were determined under which maximum survival of murine hematopoietic erythropoietin-responsive cells (ERC) could be ensured during manipulations required for cryopreservation. Cell survival was similar over freezing rates between 2 and 10 °C/min. Optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum; the DMSO was removed by centrifugation after stepwise dilution with 20 vol of medium over a 10-min period. Differing thawing rates for the cell suspensions had minimal effects on survival. “Seeding” the cell suspensions with ice crystals had no effect on ERC recovery. Overall ERC survival varied between 20 and 40%. These results confirm earlier reports that certain ERC populations are more sensitive to damage during cryopreservation than are other hematopoietic progenitor cells.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

The induction of freezing tolerance in jack pine seedlings: The role of root plasma membrane H+- ATPase and redox activities

The acquired freezing tolerance of jack pine seedlings (Pinus banksiana Lamb.) conditioned at low nonfreezing temperatures and short photoperiods was determined by comparison of seedling survival to that of nonconditioned (control) seedlings following exposure to ?5 and ?10°C. Compared to that of controls, survival of conditioned seedlings was markedly increased following exposure to freezing temperatures. A 1-week conditioning treatment significantly increased the survival of the seedlings after exposure to ?5°C, but was less effective on seedlings exposed to ?10°C. Conditioning periods of 2 and 4 weeks resulted in higher survival of seedlings exposed to both ?5 and ?10°C. The changes of two root-plasma-membrane-associated enzyme activities, H⁺-ATPase and NADH-dependent ferricyanide reductase, were studied in enriched plasma membrane fractions during conditioning and after freezing. Post-freezing activities of both enzymes were enhanced by conditioning at low temperatures and short photoperiods. These changes may be related to the increased frost hardiness also induced by conditioning.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

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.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.