Postfreeze viability of human renal epithelial carcinoma cells in quaternary solutions

The postfreeze survival of human renal epithelial carcinoma cells frozen in suspensions based on (MEM + FCS) (Eagle's Minimum Essential Medium with Hanks' salts added and supplemented with 20 vol% heat-inactivated fetal calf serum) to which additions of NaCl and the cryoprotective compounds, dimethyl sulfoxide and glycerol, were made, have been determined by a vital dye exclusion technique. A significant range of aqueous-rich quaternary solutions were found to be highly effective cryoprotective agents for these cells under optimum freezing and thawing conditions.High survival occurs in a composition series emanating from the region of high postfreeze cell viability in the ternary (MEM + FCS)-NaCl-DMSO system, between 80 and 95% (MEM + FCS), along isopleths that substitute glycerol for NaCl in various proportions. In addition, viability remains sufficiently high as a few percentage of glycerol is substituted for DMSO alone.     

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms

Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.     

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.     

Phase diagram relationships in cryobiology

The reactions which occur during freezing in biological systems employing DMSO as a cryoprotective agent may well involve information given by a near equilibrium ternary H₂O-DMSO-NaCl phase diagram. The initial freezing point depressions for solutions with three different DMSO-NaCl initial ratios (R) have been determined over the onefold surface of ice saturation. DMSO has been shown to be more effective in reducing NaCl concentration in the residual liquid than had been previously predicted. The temperature and the fraction solid which must be reached for the occurrence of second phase coprecipitation with ice have been shown to be a strong function of initial R value. Ternary invariant reactions have been identified at ?35 °C, and tentatively identified at ?115 and ?105 °C for solutions having DMSO/NaCl ratios of R = 9, 5, and 1, respectively. Metastable nonequilibrium phase formation has been observed for slow cooling of a solution with R = 1. This metastable condition results in different phase relationships upon thawing than upon the initial freezing. By quenching the system after partial rewarming, it has been demonstrated that this metastable condition can be eliminated.     

Survival of cultured cells in the cold : A kinetic study with special reference to the effect of serum in culture media

The survival at 4 °C of mouse fibroblasts (strain L-929) and rat liver cells (strain JTC-25·P5) was kinetically analysed after they had been pre-incubated at 37 °C in medium with or without supplement of serum. Both the composition of medium used for preincubation at 37 °C and that employed for storage at 4 °C had influence on the survival period.When the cells had been grown at 37 °C in Eagle minimal essential medium (MEM) alone, they rapidly lost their viability at 4 °C from the beginning. However, when grown at 37 °C in MEM supplemented with calf serum, they maintained viability at 4 °C for about 16 days and 8 days for L cells and JTC-25·P5 cells respectively, before the initiation of rapid loss of viability. The presence of macromolecular fraction of calf serum in the medium during preincubation was found to be responsible for the prolongation of survival at 4 °C.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

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

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

Effect of thawing regimens on the morphofunctional state of canine spermatozoa

The effect of 2 thawing regimens (37 degrees C for 8 sec and 55 degrees C for 5 sec) was followed up on semen parameters related to the viability of canine spermatozoa. The ejaculates were frozen in the form of pellets on dry ice in the following cryoprotective extenders: TRIS-fructose (TF), TRIS-glucose (TG), and sucrose-lactose (SL). For the 3 extenders, significant differences were found in the percentage of motile spermatozoa and their survival rate up to 300 min in favor of the 55 degrees C vs the 37 degrees C thawing regimens. Structural changes such as swelling, breakage and absence of acrosomes were observed in the samples frozen in the 3 cryoprotective extenders. A considerably lower percentage of spermatozoa with damaged acrosomes was recorded at 55 degrees C in comparison with that found at 37 degrees C (P < 0.05 for TG, TF and SL). Enzymocytochemical analysis was made of NADH-tetrazolium reductase activity in thawed spermatozoa. Cells showing moderate and strong intensity of the cytochemical reaction were found after both regimens of thawing. The percentage of spermatozoa manifesting strong intensity of the reaction was comparatively higher after thawing at 55 degrees C (31.8 +/- 2.06) than at 37 degrees C (23.7 +/- 1.41; P < 0.01). The thawing regimens were the factors that exerted influence on the morphofunctional state of frozen canine spermatozoa, irrespective of the cryoprotective extenders used, in the present study. Thus the optimal preservation of sperm viability was achieved by thawing at 55 degrees C for 5 sec.     

Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells

Normal mouse marrow cells were frozen in an automatically controlled freezer at a cooling rate of 1 °C/min to ?40 °C and 7 °C/ min to ?100 °C using dimethylsulfoxide as a cryoprotective agent. The freezing solution contained in addition either 10% homologous serum or 10% fetal calf serum. Control samples were frozen with serum-free medium. After thawing, stepwise dilution, and washing, the cells were counted, checked for CFU-s content, and cultured in Millipore diffusion chambers for 2 and 7 days.HS resulted in a recovery of 59.7% nucleated cells and 100.5% CFU-s whereas FCS and serum-free medium resulted in 59.8 and 34.7% nucleated cells and 24.5 and 18.2% CFU-s, respectively. After 2 days of culture, D.C. data showed a correlation with the CFU-s results. After 7 days of culture, no significant difference was observed between the three groups. The results of these experiments indicate that HS is required for an optimal stem cell cryopreservation and that a 2-day D.C. culture is a reliable assay system for transplantable hemopoietic tissue.     

Equilibrium and nonequilibrium phase transformations of quaternary solutions based on a physiologic support medium containing NaCl, dimethyl sulfoxide, and glycerol

Insight into the circumstances attending the freezing and thawing of biological materials suspended in cryoprotective solutions might be expected to result from knowledge of the colligative properties and glass behavior in systems based on typical physiological media, NaCl, and important cryoprotective agents. Differential thermal analysis has been used to determine phase diagram relationships in the aqueous-rich region of the quaternary system composed of a complex physiological support medium (Eagle's minimum essential medium), NaCl, and two cryoprotective compounds (glycerol and dimethyl sulfoxide). Thermograms revealed behavior corresponding to the primary crystallization of ice from sample solutions as well as the glass transition and devitrification of nonequilibrium amorphous phases. In most quaternary solutions, equilibrium-phase formation in the form of a pseudoeutectic transformation was inhibited and the formation of a metastable amorphous phase was observed as the final mode of solidification.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.

     

Protocols for thawing and cryoprotectant dilution of heart valves

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.     

Corneal cryopreservation with dextran.

Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37 degrees C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1 degrees C/min, and thawed at 37 degrees C incurred the lowest cell losses (22.4%, SD +/- 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.     

Cryopreservation in liquid nitrogen of cultured navel organe (Citrus sinensis Osb.) nucellar cells and subsequent plant regeneration

Suspension cultured cells of nucellar callus of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved. The nucellar cells were cryoprotected in Murashige-Tucker basal medium supplemented with 5% DMSO+1.2 M sucrose in an ice bath for 1 h, and then were frozen in this solution at a cooling rate of 0.5°C/min to –40°C prior to immersion in LN₂. After rapid thawing in a +40°C water bath, regrowth was achieved by transferring the treated cells, without washing, onto filter paper discs over nutrient media solidified with agar. The viability after thawing, as evaluated by FDA and phenosafranine double staining, was about 70% of controls. The revived cells resumed growth within 3 days and produced cotyledonary embryos that developed into plants within 2 to 6 months of culture. Plants regenerated from cryopreserved cells were morphologically uniform and had the characteristics typical of navel orange.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - LN₂ liquid nitrogen - NAA -naphthaleneacetic acid - SE standard error     

Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Abstract

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at ?196?°C for 24?h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p?<?0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p?<?0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Cell-mediated lymphocytotoxicity following in vitro sensitization of frozen stored human mononuclear cells in heterologous serum

Human peripheral mononuclear cells were subjected to controlled-rate freezing and stored at−196 °C. Following rapid thawing and slow removal of DMSO, the cells exhibited normal immune responsiveness to phytohemagglutinin and in mixed lymphocyte culture. After in vitro sensitization to frozen allogeneic cells in heterologous serum they developed cell-mediated lymphocytotoxicity demonstrated by ⁵¹Crrelease from specific targets isogeneic with the sensitizing cells.     

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 λunits/g liver at 37°C for 20 λmin) for isolation of intact cells for subsequent comet assay. We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation ( -85°C for 72 λh). To minimize this damage, minced liver (1.0 λg) and kidney (0.5 λg) tissues were added to 20 λml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO ( -85°C for 72 λh and 3 weeks), and to a slightly lesser extent with glycerol (72 λh), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150 λunits/g liver and 300 λunits/g kidney for 10 λmin produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at -85°C for 72 λh. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.