Isolation and low temperature preservation of adult rat heart cells

Adult rat heart cells were isolated by perfusion of the coronary system of the heart with a 0.05% collagenase solution.In one method (A), cells were finally isolated by shaking the heart fragments in a collagenase solution, after which the cells were washed and suspended in a Ca- and Mg-free buffered salt solution. The effect of different DMSO concentrations, 5, 10, 15, 20, 25, and 30% and the effect of the addition and dilution rate of DMSO on the number of trypan blue-excluding, intact, and contracting cells were studied. The highest DMSO concentration which was tolerated by the isolated adult heart cells was 15%. Variation of addition rate and the dilution rate of DMSO had no effect. After freezing at external cooling rates of 1, 5, 10, 30, and 50 °C/min to ?100 °C, and then rapidly to ?196 °C, in the presence of 5, 10, or 15% DMSO, reanimation of these cells was not achieved.In another method (B), heart fragments, after collagenase perfusion of the heart, were first treated with 5, 10, or 15% DMSO, after which the cells were isolated. If these cells were frozen at 1 °C/min with 10% DMSO, 15% of the cells, expressed as a percentage of the control, remained morphologically intact and 38% of the cells were contracting after thawing. Significantly higher survival percentages of 30 and 61%, respectively, were obtained if the heart fragments were left intact during freezing.     

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.     

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.     

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.     

The effect of cooling rate and of dimethyl sulfoxide concentration on the ultrastructure of neonatal rat heart cells after freezing and thawing

The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the survival in culture.Without freezing, increasing DMSO concentrations caused an increase of morphological damage, correlating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nuclei with clumped chromatin at 10% DMSO.After freezing at optimal or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cooling rates and DMSO concentrations appeared to have a normal structure.     

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.     

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.     

Cryopreservation of human bone marrow‐derived mesenchymal stem cells with reduced dimethylsulfoxide and well‐defined freezing solutions

The aim of this study is to investigate the feasibility of using well defined, serum‐free freezing solutions with a reduced level of dimethylsulfoxide (DMSO) of 7.5, 5, and 2.5% (v/v) in the combination with polyethylene glycol (PEG) or trehalose to cryopreserve human bone marrow‐derived mesenchymal stem cells (hBMSCs), a main source of stem cells for cell therapy and tissue engineering. The standard laboratory freezing protocol of around 1°C/min was used in the experiments. The efficiency of 1,2‐propandiol on cryopreservation of hBMSCs was explored. We measured the post‐thawing cell viability and early apoptotic behaviors, cell metabolic activities, and growth dynamics. Cell morphology and osteogenic, adipogenic and chondrogenic differentiation capability were also tested after cryopreservation. The results showed that post‐thawing viability of hBMSCs in 7.5% DMSO (v/v), 2.5% PEG (w/v), and 2% bovine serum albumin (BSA) (w/v) was comparable with that obtained in conventional 10% DMSO, that is, 82.9 ± 4.3% and 82.7 ± 3.7%, respectively. In addition, 5% DMSO (v/v) with 5% PEG (w/v) and 7.5% 1,2‐propandiol (v/v) with 2.5% PEG (w/v) can provide good protection to hBMSCs when 2% albumin (w/v) is present. Enhanced cell viability was observed with the addition of albumin to all tested freezing solutions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

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 a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

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 platelets using trehalose: The role of membrane phase behavior during freezing

In blood banks, platelets are stored at 20–24°C, which limits the maximum time they can be stored. Platelets are chilling sensitive, and they activate when stored at temperatures below 20°C. Cryopreservation could serve as an alternative method for long term storage of platelet concentrates. Recovery rates using dimethyl sulfoxide (DMSO) as cryoprotective agent, however, are low, and removal of DMSO is required before transfusion. In this study, we have explored the use of trehalose for cryopreservation of human platelets while using different cooling rates. Recovery of membrane intact cells and the percentage of nonactivated platelets were used as a measure for survival. In all cases, survival was optimal at intermediate cooling rates of 20°C min^?1. Cryopreservation using DMSO resulted in high percentages of activated platelets; namely 54% of the recovered 94%. When using trehalose, 98% of the platelets had intact membranes after freezing and thawing, whereas 76% were not activated. Using Fourier transform infrared spectroscopy, subzero membrane phase behavior of platelets has been studied in the presence of trehalose and DMSO. Furthermore, membrane hydraulic permeability parameters were derived from these data to predict the cell volume response during cooling. Both trehalose and DMSO decrease the activation energy for subzero water transport across cellular membranes. Platelets display a distinct lyotropic membrane phase transition during freezing, irrespective of the presence of cryoprotective agents. We suggest that concomitant uptake of trehalose during freezing could explain the increased survival of platelets cryopreserved with trehalose. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws

The effect of varying the concentration of glycerol from 0 to 16% on the survival of ram spermatozoa frozen at increasing rates of cooling (1–100 °C/min) or by direct plunging of spermatozoa in 0.5-ml straws in liquid nitrogen was studied after thawing at a constant rate (in water at 39 °C for 30 sec). For each glycerol concentration, the ram spermatozoa tolerated a range of cooling velocities and the best survival rates (percentage motility and rating) were obtained when the glycerol concentration was 4 or 6% and when the rate of freezing ranged from 10 to 100 °C/min. No spermatozoa survived in any glycerol concentration following freezing in straws plunged into liquid nitrogen. In general, the range of cooling rates shifts to lower values as the glycerol concentration increases for optimum cryosurvival. However, the toxic effect of increasing the concentration of glycerol over 8% contributes greatly to the gradual decrease in cryosurvival of spermatozoa at these particular concentrations.     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

Freezing mammalian embryos: A review of the techniques

This article reviews the literature on freezing mammalian oocytes and embryos, with emphasis on embryos of domestic animals. Mammalian embryos must be stored in a quiescent state to retain viability for long periods. This has been accomplished by freezing and storing the embryos at ?196°C. To freeze embryos, a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol was required, slow cooling (0.1 to 2.0°C/min) and warming (1 to 50°C/min) rates were used, enucleation or seeding the freezing medium was a necessity, and stepwise addition and removal of the cryoprotectant at room temperature seemed to be beneficial. Using the above parameters embryos have been frozen and stored at ?196°C for several years and upon thawing and transfer to a suitable recipient, viable offspring have developed. Initially embryo viability was low after freezing-thawing, but with refinement of freezing-thawing techniques has increased sufficiently so that freezing embryos is no longer a laboratory technique, but is applicable to field use.     

Bioassay to measure effects of cooling and warming rates and protection by dimethyl sulphoxide on survival of frozen Babesia rodhaini

The percentages of Babesia rodhaini parasites that survived different rates of cooling to −79 °C were determined by titrating infectivity in CBA mice before freezing and after thawing. The cryoprotective effect of DMSO and the effect of warming rate were also assessed.When parasitized blood containing 1.5 DMSO was cooled at nominal rates of 2.5 °, 265 °, and 2785 °C/min and warmed at 4320 °C/min, the respective survival rates were 0.075, 4.9, and 0.1%, indicating the existence of an optimal cooling rate. Blood without DMSO cooled and warmed under the same conditions was over 1000 times less infective. When parasitized blood containing DMSO was cooled at 2785 °C/min and warmed at 4320 °, 24.5 °, and 1.84 °C/ min, infectivity decreased progressively with the warming rate. The degrees of haemolysis in frozen and thawed blood indicated that cooling rate was more important than an intact host cell to survival of the parasite.The growth rate of B. rodhaini in CBA mice, estimated to be one binary fission in 8.5 hr, was not affected by the addition of DMSO followed by freezing and thawing.