The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.     

Frequency and coordination of ciliary beat after cryopreservation of respiratory epithelium

The effect of cryopreservation on human nasal mucosal biopsies was evaluated by determining the frequency and coordination of the ciliary beat. Samples were cryopreserved in a medium containing 80% Gey's balanced salt solution, 10% dimethyl sulfoxide, and 10% fetal calf serum. After thawing, the samples were put in a solution of 90% Gey's balanced salt solution and 10% fetal calf serum. Video recordings of the samples before and after cryopreservation were compared using a semiquantitative method. All the frequencies and coordination patterns seen before cryopreservation could be found in the sample after cryopreservation. It is concluded that ciliated epithelial biopsies can be stored in liquid nitrogen with the maintenance of ciliary beat frequency. In the recorded ciliated cells the ciliary beat coordination was slightly reduced; a lack of coordination was present in 20% of cells after cryopreservation as compared to 10% before cryopreservation.     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Effects of Additives on the Survival of Lactic Streptococci in Frozen Storage

Three single-strain cultures, Streptococcus lactis C₂, S. cremoris R₁, and S. diacetilactis DRC₂, were frozen and stored in skim milk, in skim milk containing apple juice, and in skim milk containing one of the following additives: glycerol (10%, v/v), dimethyl sulfoxide (10%, v/v), l-malic acid (0.5 and 2.0%, w/v), acetamide (0.5 and 2.0%, w/v), or succinimide (0.5 and 2.0%, w/v). Cultures were frozen and stored at -23.3 C, frozen and stored at -196 C in liquid nitrogen, or frozen at -196 C and stored at -23.3 C. Cultures frozen and stored at -196 C in liquid nitrogen gave the greatest recovery of viable cells. The number of cells surviving after storage at -23.3 C was greater when the cells had been frozen in liquid N₂ than when they had been frozen at -23.3 C. All strains stored at -23.3 C showed a decrease in numbers of surviving cells; additives, particularly l-malic acid and apple juice, were advantageous in preserving the viability of the S. lactis C₂ and S. cremoris R₁ strains, but had little or no effect on the survival of S. diacetilactis DRC₂. l-Malic acid and apple juice stimulated acid production for all cultures in activity tests following incubation after thawing, whereas glycerol and dimethyl sulfoxide retarded its development.     

Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid

Abstract The cryopreservation of an anaerobic rumen fungus, Piromyces communis OTS1, was examined at −84 °C using dimethyl sulfoxide, propylene glycol or ethylene glycol as cryoprotectants. Ethylene glycol was the most effective agent, combining high survival and low toxicity, followed by dimethyl sulfoxide and propylene glycol. Cell-free rumen fluid in the cryopreservation medium decreased the toxicity of the cryoprotectant agents and also had a protective action per se. A survival of 80% after 1 year storage was obtained when samples with an initial zoospore density of 5 × 10⁴ zoospores/ml were equilibrated for 15 min in medium containing 0.64 M ethylene glycol and 5% cell-free rumen fluid, then frozen with dry ice and stored at −84 °C.     

Viability and Infectivity of Measles Virus-Infected Nervous Tissue Frozen in Cryoprotective Medium

Measles virus-infected hamster brain tissue remained viable when frozen slowly in medium containing dimethyl sulfoxide. This procedure was essential for isolation of neurotropic measles virus from tissue specimens stored frozen.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol-12-myristate-13-acetate

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Exposure to sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristine-13-acetate alters sensitivity of K562 cells to nonspecific lysis by human or rat leukocytes

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to nonspecific lysis (nonrestricted with respect to antigens of the major histocompatibilty complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Biosynthesis of spectrin and its assembly into the cytoskeletal system of Friend erythroleukemia cells

Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.     

Cryopreservation of isolated blood vessels

Canine saphenous veins were immersed in fetal calf serum (FCS) containing various cryoprotective agents, slowly frozen and stored for several weeks at subzero temperatures. Pharmacological investigations of frozen/thawed tissues revealed considerable attenuation of the contractile force of frozen stored veins as compared to unfrozen veins. The best recovery after thawing of frozen stored canine veins was obtained on tissues which had been frozen slowly to -70 degrees C and stored in liquid nitrogen while being immersed in FCS containing 1.8 mol/l dimethyl sulfoxide (DMSO). Though the maximum response to noradranline of helical strips prepared from these veins was diminished to about 60% the evidence suggests that there may be a very good preservation of the main biochemical properties, such as monoamine oxidase activity, endogenous prostaglandin synthesis and neuronal uptake mechanism in veins stored under these conditions. The same method of cryopreservation was applied to store samples of human veins. Comparison of the pD2 values for various agonists and of the blocking activities of various antagonists of both 5-HT receptors and alpha-adrenoceptors yielded an excellent correlation between the parameters determined on frozen/thawed and unfrozen human veins. It is concluded that freezing isolated blood vessels may be considered an effective means of preserving and storing vascular tissues for pharmacological investigations.     

Stimulation of plasminogen activator production by dimethyl sulfoxide in Chinese hamster ovary cells

The production of plasminogen activator activity in an auxotrophic mutant of the Chinese hamster ovary cell line was found be greatly stimulated by low concentrations of dimethyl sulfoxide. The production of both cell-associated and excreted plasminogen activator activities was stimulated maximally by dimethyl sulfoxide at a concentration of 2.5%. The stimulation of plasminogen activator activity production was found to be completely inhibited by actinomycin D and cycloheximide but not by mitomycin C, implying that new protein and RNA syntheses were required for this process. Using specific antibodies against plasminogen activator, the presence of a tissue-type plasminogen activator could only be detected in dimethyl sulfoxide treated cells. The dimethyl sulfoxide induced plasminogen activator production was observed only in a mutant auxotrophic for adenosine, glycine, and thymidine but not in wild-type cells. The ability of dimethyl sulfoxide to induce the synthesis of plasminogen activator was lost when the cells were hybridized with another complementary auxotrophic mutant. This implies that the ability of dimethyl sulfoxide to stimulate the production of plasminogen activator may be related to the auxotrophic mutation in this cell.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. II. Protection from lethal, chromosomal, and DNA damage

Golden hamster embryo cells were exposed to 137Cs gamma rays in the presence or absence of dimethyl sulfoxide at both 310 and 77 K. Dimethyl sulfoxide gave significant protection against cell killing at both 310 and 77 K. The extent of radioprotection with 1.28 M dimethyl sulfoxide at 77 K was 85-89% of the lethal effects observed in the absence of dimethyl sulfoxide at 310 K; the dose-modifying factor was 5.7. Dimethyl sulfoxide also exerted protected against gamma-ray-induced DNA single-strand breaks and chromosomal aberrations with a maximum protection of 80-100% at a dimethyl sulfoxide concentration of 1.28 M at 77 K. At 77 K, H atoms, ion holes, and electrons can migrate through frozen cells but OH radicals cannot diffuse. Thus the protective effects of dimethyl sulfoxide against cell killing, chromosomal aberrations, and DNA single-strand breaks at 77 K may be due to the scavenging of H atoms or other ions, rather than OH radicals.     

The serial cultivation of suspended BHK-21/13 cells in serum-free Waymouth medium.

A simple medium system was developed to obtain growth of BHK-21 cells in shaker cultures in the absence of serum. These cells have now undergone over 80 serial passages in serum-free Waymouth medium and have been recovered from the frozen state after storage for over 1 month in medium containing 10% dimethyl sulfoxide (DMSO) and 1% bovine serum albumin (BSA). Various amounts of exogenous lipid in the form of sodium oleate were added to cultures of cells growing in serum-free Waymouth medium. Concentrations of 10-50 mug of sodium oleate/ml had no detrimental effects on the cells as measured by trypan blue uptake. Furthermore, the cells were serially passed ten times in the presence of 10 mug sodium oleate/ml. Depletion of calf serum from the growth medium and addition of known quantities of lipids to the system provides a means of revealing subtle changes in lipid synthesis and lipid turnover during cellular growth.     

Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol.

A solvolytic N-desulfation of heparin was developed by treatment of its pyridinium salt with dimethyl sulfoxide containing 5% of water or methanol for 1.5 h at 50 degrees. Chemical and chromatographic studies showed that the solvolytic desulfation is a useful method for N-desulfation of heparin without depolymerization of the heparin molecule. The partially N-desulfated heparins were also obtained by treatment with dimethyl sulfoxide containing 5% of water at 20 degrees, and their anticoagulant activity is related to the degree of N-desulfation.     

Freezing of rat macrophages

The effect of freeze-thawing was studied in cryoprotected (10% dimethyl sulfoxide) rat peritoneal macrophages. Recovery after post-thaw washing was about 50%. Phagocytosis of latex particles seemed unhampered by this procedure, whereas the abilty to adhere to glass and the amount of Fc and C₃ receptors were moderately reduced. Low temperature preservation of macrophages might be a useful storage method, as it is for lymphocytes and tissue culture cells.     

Cryopreservation of spermatozoa of the American oyster, Crassostrea virginica Gmelin

Spermatozoa of the American oyster Crassostrea virginica were frozen to ?196 °C in concentrated Hanks' salt solution (2.6×) containing 8% dimethyl sulfoxide. About 0.2 ml of spermatozoa that were stored in liquid nitrogen for 68 days fertilized 91% of 65,600 eggs compared to fresh spermatozoa, which fertilized 92% of approximately the same number of eggs from the same females. Larvae resulting from fertilization of fresh eggs with 39-day-old cryopreserved spermatozoa appeared normal after 11 days.     

Inhibition of insulin receptor binding by dimethyl sulfoxide

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of ¹²⁵I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of ¹²⁵I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.     

9<Superscript>th</Superscript> International Conference Plant Functioning Under Environmental Stress September 12-15, 2012 Cracow Poland

High productive suspension cultures of Genista tinctoria were elicited (methyl jasmonate or chitosan) and permeabilised (dimethyl sulfoxide) in order to achieve a plant in vitro system rich in isoflavones, with extracellular storage profile. The maximum concentration of isoflavone aglycones (4–6 times higher than in the controlled biomass) was obtained in the suspension elicited with chitosan. All isoflavonoids were stored inside cells. In case of methyl jasmonate supplementation the total concentration of isoflavone aglycones achieved was the result of the de novo biosynthesis as well as a hydrolysis of the storage ester forms. The presence of chitosan in the medium was only associated with the production of aglycones de novo. The elicitors had no effect on the accumulation and metabolism of the basic glycoside isoflavones in the suspension. The change in the way the isoflavones were stored was achieved after supplementing the growth media with dimethyl sulfoxide. Maximum concentration of isoflavones in the medium was observed in the 7th hour of the experiment. Eventually, a plant growth system was developed, producing over 11% of isoflavones, ejected as a result of chemical permeabilisation, into the growth medium (approx. 80% of total amount of these compounds in the biomass).     

Cryopreservation of Leucocytozoon caulleryi sporozoites

Leucocytozoon caulleryi sporozoites that had been stored at -196 degrees C or -80 degrees C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae, which had been stored at -80 degrees C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.