Optimization of slow cooling cryopreservation for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single‐cell dissociation. After confirming hPSC survivability after freeze‐thawing, we found that hPSCs that were freeze‐thawed as colonies showed markedly decreased survival, whereas freeze‐thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze‐thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post‐thawing. The improved method was also adapted to a xeno‐free culture system. Moreover, the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin‐521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high‐quality hPSCs. genesis 52:49–55, 2014. © 2013 Wiley Periodicals, Inc.     

Exothermic responses of dormant Salix stems during exposure to subzero temperatures

Differential thermal analysis (DTA) was used to determine the exothermic responses in dormant stems and excised lengths of stem of Salix dasyclados Wimmer subjected to artificial freezing treatments.
The presence of ice on the surfaces of intact stems restricted the mechanism of freezing avoidance to temperatures above –4°C. In contrast, excised lengths of stem started to freeze as soon as the ambient temperature fell below –2°C, demonstrating that extracellular ice formation takes place earlier if cut surfaces are present. Exposure of dormant excised lengths of stem to subfreezing temperatures for more than 8 weeks did not alter their nucleation temperature not their exothermic differential responses. Early extracellular crystallisation of freezable cellular water provides conditions that allow dormant Salix dasyclados stems or excised lengths of stem to survive extreme freezing stress.
Crystallisation of extracellular and cellular water took place in the cortex, and did not result in visual damage or reduced survival. This nucleation of extracellular water took place over the same temperature range whether the excised dormant lengths of stem were partly (bark only) or completely thawed. Exposure of dormant tissue to 20°C for up to 24 h did not alter the level of freezing tolerance, nor did it increase the susceptibility of excised lengths of stem to damage by extreme temperature fluctuations.     

Effect of varying freezing and thawing rates in experimental cryosurgery

Six different freezing/thawing programs, which varied freezing rate, duration of freezing, and thawing rates, were used to investigate the effect of these factors on cell destruction in dog skin. The range of tissue temperatures produced was from -15 to -50 degrees C. The extent of destruction was evaluated by skin biopsies 3 days after cold injury. In single, short freezing/thawing cycles, the temperature reached in the tissue was the prime factor in cell death. Longer freezing time and slow thawing were also important lethal factors which increased destruction of cells. Cooling rate, whether slow or fast, made little difference in the outcome. The experiments suggested that present-day, commonly employed cryosurgical techniques, which feature fast cooling, slow thawing, and repetition of the freeze/thaw cycle, should be modified by the use of maintenance of the tissue in the frozen state for several minutes and slow thawing. Thawing should be complete before freezing is repeated. These modifications in technique will maximize tissue destruction, an important consideration in cancer cryosurgery.     

Optimal conditions for freezing CHO-S and HEK293-EBNA cell lines: influence of Me2SO, freeze density, and PEI-mediated transfection on revitalization and growth of cells, and expression of recombinant protein

To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

TWO SPECIFIC CAUSES OF CELL MORTALITY IN FREEZE‐THAW CYCLE OF YOUNG THALLI OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Porphyra yezoensis Ueda is an important marine aquaculture crop with single‐layered gametophytic thalli. In this work, the influences of thallus dehydration level, cold‐preservation (freezing) time, and thawing temperature on the photosynthetic recovery of young P. yezoensis thalli were investigated employing an imaging pulse‐amplitude‐modulation (PAM) fluorometer. The results showed that after 40 d of frozen storage when performing thallus thawing under 10°C, the water content of the thalli showed obvious effects on the photosynthetic recovery of the frozen thalli. The thalli with absolute water content (AWC) of 10%–40% manifested obvious superiority compared to the thalli with other AWCs, while the thalli thawed at 20°C showed very high survival rate (93.10%) and no obvious correlation between thallus AWCs and thallus viabilities. These results indicated that inappropriate thallus water content contributed to the cell damage during the freeze‐thaw cycle and that proper thawing temperature is very crucial. Therefore, AWC between 10% and 40% is the suitable thallus water content range for frozen storage, and the thawing process should be as short as possible. However, it is also shown that for short‐term cold storage the Porphyra thallus water content also showed no obvious effect on the photosynthetic recovery of the thalli, and the survival rate was extremely high (100%). These results indicated that freezing time is also a paramount contributor of the cell damage during the freeze‐thaw cycle. Therefore, the frozen nets should be used as soon as time permits.     

Multiple freezing points as a test for viability of plant stems in the determination of frost hardiness

A technique is presented for a simple, rapid, and reliable means of determining the viability of plant tissue subjected to freezing temperatures. Freezing curves of excised stems of Cornus stolonifera Michx., and several other genera were studied. Tissue temperature was recorded during freezing of plant stem sections. The heat of crystallization deflected the resultant freezing curves at points where tissue froze. Living stem sections of all genera studied revealed 2 freezing points, while dead tissue exhibited only 1. The influence of variables such as moisture content, sample size, thermocouple placement, and cooling rate on freezing curves was analyzed. Stem samples wrapped in moisture-proof film with a thermocouple inserted into the pith were frozen to a predetermined test temperature, thawed, and subjected to a second freezing cycle. The presence or absence of 2 freezing points in the second freezing cycle was used as a criterion for establishing viability. The results were immediately available and identical to results from regrowth tests which took about 20 days.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Evaluating of influence of repeated thaw/freeze cycles on IgG and IgM stability

The purpose of this study was evaluating influence of repeated cycles of thawing and freezing serum samples on IgG and IgM stability. Serum positive samples for anti-cytomegalovirus and antimeasles virus IgG and/or IgM were aliquoted. Tubes containing 100 microl aliquot were frozen at -20 degrees C. Samples were repetitively thawed and froze in one to ten cycles. Antibodies presence was examined with commercially available ELISA tests. All samples reminded positive even after ten repeated thaw/freeze cycles.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

Frost fatigue response to simulated frost drought using a centrifuge in Acer mono Maxim

In the coldest part of winter, water uptake is blocked by the frozen soil and frozen stems known as ‘frost drought’ causing severe embolisms in woody plants. Frost drought in stems was simulated in a centrifuge by a synergy between freeze–thaw cycles and the different tensions induced by changing the rotation speed. Frost fatigue was defined as a reduction of embolism resistance after a freeze–thaw cycle and determined from ‘vulnerability curves’, which showed percent losses of conductivity vs tension (positive value) or xylem pressure (negative value). Different tensions combined with a controlled freeze–thaw cycle were induced to investigate the effects on frost resistance over the course of year. During the growing season, Acer mono Maxim. developed significant frost fatigue, and a significant positive correlation was found between frost fatigue response and exogenous tension. During the dormant season, A. mono showed very high embolism resistance to frost drought, even under a tension of 2 MPa. When the exogenous tension was increased to 3 MPa while the stem was frozen, significant frost fatigue occurred. Longer freezing times had more serious effects on frost fatigue in A. mono. A hypothesis was raised that at the same low temperature, the severer the drought (higher tension) when stems were frozen, the higher frost fatigue response would be induced.     

A requirement for sucrose in xylem sap flow from dormant maple trees

The response of excised stem segments of several tree species to freezing and thawing cycles was studied. All species studied (Thuja occidentalis, Fagus grandifolia, and Betula papyrifera) except maple (Acer spp.) exuded sap while freezing and absorbed on thawing. Maple stems absorbed sap while freezing and exuded sap during the thaw only when sucrose was present in the vessel solution. Increased concentration of sucrose in the vessel sap led to increased exudation. In the absence of sucrose, maple stems absorbed sap on thawing. The presence of sucrose enhanced sap absorption during freezing cycles in maples. In general, large sugars, disaccharides and larger, could substitute for sucrose in the maple exudation response while sugar hexoses could not. The results are discussed in relation to the O'Malley-Milburn model (1983 Can J Bot 61: 3100-3106) of sap flow in maples.     

Primmorphs Cryopreservation: A New Method for Long-Time Storage of Sponge Cells

The possibility to cryopreserve cells allows for wide opportunities of flexible handling of cell cultures from different sponge species. Primmorphs model, a multicellular 3D aggregate formed by dissociated sponge cells, is considered one of the best approaches to establish sponge cell culture but, in spite of the available protocols for freezing sponge cells, there is no information regarding the ability of the latter to form primmorphs after thawing. In the present work, we demonstrate that, after a freezing and thawing cycle using dissociated Petrosia ficiformis cells as a model, cells viability was high but it was not possible to obtain primmorphs. The same protocol for cryopreservation was then used to directly freeze primmorphs. In this second case, after thawing, viability and the cellular proliferative level were similar to unfrozen standard primmorphs. Spiculogenesis in thawed primmorphs was evaluated by quantifying the silicatein gene expression level and by assaying the silica amount in the newly formed spicules, then compared with the correspondent values obtained in standard unfrozen primmorphs. Results indicate that the freezing cycle does not affect the spiculogenesis rate. Finally, the expression level of heat shock protein 70, a well-known stress marker, was assayed and the results showed no differences between frozen and unfrozen samples. These findings are likely to promote relevant improvements in sponge cell culture technique, allowing for a worldwide exchange of living biological material, paving the way for cell banking of Porifera.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: Influence of warming rate

The general belief is that slow freezing injury is either the result of exposure to high salt concentrations or the result of excessive cell shrinkage. Increased salt concentration arises as increasing amounts of pure ice precipitate out of solution during freezing and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. Cell shrinkage is an osmotic response to the concentration of external solutes. The consensus has been that the injury is related to the composition of the solution in these channels and not to the amount of residual liquid.Ordinarily, salt concentration and the amount of liquid in the unfrozen channels are reciprocally related; but they can be separated within limits by varying the total concentration of solutes in the suspending medium while holding the mass ratio of additive to salt constant, and by then slowly freezing samples to various subzero temperatures, chosen to produce various molalities of salt, while holding the unfrozen fraction constant, or vice versa. We have recently reported (9) that when human red cells are frozen under these conditions and thawed rapidly, survival is more dependent on the unfrozen water fraction than it is on the salt concentration in that fraction. The present work compares these results with those obtained with slow thawing. While the general conclusion remains unaltered, slowly thawed cells were able to survive the freezing of a higher fraction of extracellular water than were rapidly thawed cells.Calculations were made of the changes in cell volume during the equilibration with glycerol and the subsequent freezing involved in these experiments. Cell size and cell solute concentration were found to be independent of the fraction of unfrozen extracellular water, but cell survival was strongly dependent on that fraction. If applicable to other than human red cells, this finding is likely to require major modifications in current views of slow-freezing injury and its prevention.     

A successful technique for the preservation of rabbit embryos

A technique for successfully freezing, thawing and transferring rabbit embryos has been developed. Morula stage embryos were collected from super-ovulated female rabbits by flushing both oviducts and uterine horns with a tissue culture medium. Well developed, viable embryos were then transferred to freezing vials and a cryoprotectant, dimethyl sulfoxide (DMSO) was added in several steps to bring its final concentration to 1.6 molar. To freeze the embryos the temperature was lowered slowly (either 0.5 degrees C/min or 1.0 degrees C/min) to -80 degrees C at which point the vials were transferred directly to liquid nitrogen (-196 degrees C). Thawing was done at 8 degrees C/min. After thawing, phosphate buffered saline was added in a stepwise manner to dilute the DMSO. The thawed embryos were then cultured at 37 degrees C. Transfer of the embryos was accomplished by laparotomizing a pseudopregnant doe and introducing the embryos into the fimbriated ends of the oviducts. The 101 positively transferred embryos resulted in 45 implantations and 34 live born young.     

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching −40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.