The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Effects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membranes and mitochondria to striped bass (Morone saxatilis) sperm

Intact plasma membrane and functional mitochondria are important attributes for the fertilization capacity of fish sperm. In the present study, dimethyl sulfoxide (Me(2)SO) and glycine were investigated in an effort to improve plasma membrane integrity and mitochondrial function in cryopreserved striped bass (Morone saxatilis) sperm. Prior to freezing, no concentration of Me(2)SO (2.5, 5 or 10%) was found to affect (P>0.05) the integrity of plasma membranes after sperm were exposed for 10 min. However, mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. Both fluorescent staining and microscopic examination of the ultrastructure of post-thaw plasma membranes indicated that with increasing Me(2)SO concentration, plasma membranes were better protected, and 10% Me(2)SO had the highest percentage of sperm with plasma membranes intact. However, sperm mitochondrial function decreased (P>0.05) with increasing Me(2)SO concentration. The inverse relationship between plasma membrane integrity and mitochondrial function, given the Me(2)SO concentration, suggests that care must be taken to select Me(2)SO concentration that will maximize the protection of both plasma membranes and mitochondrial function. The addition of glycine to the cryomedia increased (P<0.05) the percentage of sperm with post-thaw functional mitochondria and ATP content. However glycine did not provide (P<0.05) protection to post-thaw plasma membrane integrity. The highest percentage of sperm with both intact plasma membranes and functional mitochondria was obtained with 7.5% Me(2)SO and 75 mM glycine.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.     

Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.     

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

Cryopreservation in micro-volumes: impact upon Caco-2 colon adenocarcinoma cell proliferation and differentiation

Recent advances in cell-based therapies require new approaches for cell cryopreservation, capable of dealing with large number of samples and providing specific conditions for each cell type. Reduction of sample volume from the commonly used 1 mL to 25 microL in 30-well micro-cryosubstrates improves cryopreservation by allowing automation, data handling and access to individual wells without thawing the whole cryosubstrate. This system was evaluated for the storage of Caco-2 colon adenocarcinoma cells, which differentiate spontaneously after long-term culture. The impact of the cryosample small volume upon post-thawing membrane integrity of the cells and their capacity to proliferate and differentiate was studied. Two different cryoprotectants commonly employed, dimethyl sulfoxide (Me(2)SO) and glycerol, were evaluated as well as the possibility of decreasing their concentration from the 10% concentration, usually used, down to 3% (v/v). The process automation by pipette robotic addition of the cryoprotectant to the micro-cryosubstrates was also evaluated. The micro-cryosubstrates have proven to be at least as efficient as typical 1 mL cryovials for cryopreservation of Caco-2 cells using either Me(2)SO or glycerol. Compared to the manual process, the automatic addition of glycerol to the micro-cryosubstrates allowed higher cell viabilities after thawing while with Me(2)SO no significant changes were observed. Me(2)SO has shown to be more effective than glycerol in maintaining high post-thaw cell membrane integrity, either in micro-cryosubstrates or cryovials, for any of the concentrations tested. The ability of Me(2)SO in maintaining high cell membrane integrity post-thawing was confirmed by long-term (up to 22 days) proliferation and differentiation studies performed with cells cultured immediately after thawing.     

Stimulatory and inhibitory effects of dimethyl sulfoxide and ethylene glycol on ATPase activity and calcium transport of sarcoplasmic membranes.

1. The effect of dimethyl sulfoxide (Me2SO) and ethylene glycol on two different preparations of the sarcoplasmic reticulum, i.e. native membranes and membranes whose phospholipids were hydrolyzed by phospholipase A, were investigated using ATP and p-nitrophenylphosphate as substrates. 2. Me2SO and ethylene glycol inhibit both calcium-dependent ATP hydrolysis and ATP-supported calcium transport by native vesicles. 3. In contrast, calcium-dependent p-nitrophenylphosphatase activity as well as p-nitrophenyl-phosphate-supported calcium transport are activated by both agents at concentrations lower than 30% (v/v). 4. Me2SO strongly stimulates p-nitrophenylphosphate activity of vesicles treated with phospholipase A, but has relatively little effect on p-nitrophenylphosphatase activity of native vesicles. 5. Up to a concentration of approximately 40% Me2SO (v/v) the inhibiting effect on the calcium-dependent ATPase is fully reversible, but only partially reversible on calcium transport. 6. In the concentration range where Me2SO inhibits ATP hydrolysis and calcium transport, it does not affect ATP binding to the membranes nor calcium-dependent formation of phospho-protein. 7. The rate of dephosphorylation as well as the rate of Pi exchange between ATP and ADP are markedly reduced by the presence of 30% Me2SO (v/v). 8. While Me2SO inhibits passive calcium efflux, ethylene glycol produces a considerable activation. 9. ADP-dependent calcium efflux and ATP synthesis are activated by 15% Me2SO (v/v). Ethylene glycol reduces both activities. 10. The results suggest that the respective substrate-enzyme complexes are differently affected by the agents, resulting either in inhibition or stimulation     

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140–170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.     

Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase

Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Conformational change in beef-heart mitochondrial F1 ATPase to ATP synthesis mode induced by dimethylsulfoxide and ATP revealed by sulfhydryl group labeling

Treatment of beef-heart mitochondrial F1 ATPase with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) results in the incorporation of 1 mol DTNB/mol F1 without loss of ATPase activity. Incorporation is not prevented by ATP. Labeling occurs predominantly on an alpha-subunit, but also with a significant degree of modification of gamma- and epsilon-subunits. It is suggested that the modified sulfhydryl groups of the alpha-, gamma- and epsilon-subunits are in proximity so that only one can be modified by the reagent. Guanidine hydrochloride (0.3 M) dissociates F1 into its subunits. Eight sulfhydryl groups/mol F1 can be modified under these conditions. Guanidine hydrochloride does not cause dissociation of F1 in the presence of 30% (v/v) dimethylsulfoxide (Me2SO) and 2 mM ATP. Under these conditions a second molecule of DTNB is incorporated into F1 with nearly equal modification of the epsilon-subunit and an alpha-subunit. It is proposed that Me2SO and ATP induce a more stable conformation of F1, which is resistant to dissociation by guanidine hydrochloride, but in which the site of reaction with DTNB is made more accessible by the guanidine hydrochloride to permit the simultaneous modification of an alpha-subunit and the epsilon-subunit. This conformation is probably that which occurs during ATP synthesis by F1 in the presence of Me2SO.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

Cryopreservation of isolated fish blastomeres: effects of cell stage, cryoprotectant concentration, and cooling rate on postthawing survival

The toxicity of the cryoprotectant dimethyl sulfoxide (Me(2)SO) to isolated blastomeres was examined in three fish species representative of distinct environments: marine (whiting, Sillago japonica); estuarine (pejerrey, Odontesthes bonariensis); and freshwater (medaka, Oryzias latipes). The effects of embryonic stage, Me(2)SO concentration, and cooling rate on the cryopreservation of blastomeres were also studied. Whiting sheds small planktonic eggs whereas the other two species shed large demersal eggs. Isolated blastomeres from the three species tolerated Me(2)SO concentrations up to 9% relatively well for over 5 h but lost viability rapidly at 18%. Cells from later embryonic stages (512 or 1024 cells) were more tolerant of Me(2)SO than those from earlier stages (128 or 256 cells). The three factors examined, alone or in combination, had a significant effect on the survival of blastomeres after freezing and thawing, but the extent of the effect and the optimum conditions varied with the species. In general, the highest rates of successful cryopreservation were observed with older rather than younger blastomeres, slower rather than faster cooling, and with 9-18% rather than 0% Me(2)SO. Survival rates for blastomeres cryopreserved under the most effective combination of the three factors examined for each species were 19.9 +/- 10.1% for whiting, 34.1 +/- 8.5% for medaka, and 67.4 +/- 12.8% for pejerrey. Copyright 1999 Academic Press.