Combination Of DMSO Research Articles

Thermally generated bromine “Cation Pool-”mediated oxidation of indole to 2-oxindole

Unlike the electrolytic generation of bromine cation pool, thermally generated bromine cation pool was employed for regioselective oxidation of indole to 2-oxindole at room temperature. A combination of DMSO, DBE, and morpholine at 80 °C generates the bromine cation pool, which in turn was utilized for bromination followed by hydroxylation by water through a dearomatization and rearomatization pathway to afford the desired 2-oxindoles. At higher temperature the in situ generated 2-oxindoles undergo α-bromination followed by DMSO-mediated Kornblum oxidation to afford isatin derivatives. This bromine cation pool strategy was further exploited for one-pot synthesis of spirooxindole under metal-free conditions.

P10.31.A OSIMERTINIB AND OTX008 IMPAIR SURVIVAL OF GLIOBLASTOMA CELLS BY INHIBITING RAS

Abstract BACKGROUND Tyrosine kinase receptors play a critical role in the progression of almost all malignancies. In 83% of glioblastoma the endothelial growth factor receptor (EGFR) is affected by genetic alterations leading to cell proliferation and therapy resistance. This leads to a constitutive activation of EGFR, following the activation of the PI3K/AKT and RAS/MAPK signalling pathways. The EGFR-inhibitor Osimertinib showed anti-tumor effect in lung cancer patients with cerebral metastasis. OTX008 is a selective inhibitor of Galectin-1, a small protein, which is involved in stabilising the active form of H-Ras and thereby enables the downstream PI3K/AKT and RAS/MAPK pathways. This study was performed to investigate whether OTX008, Osimertinib and a combination of both have an antiproliferative effect and inhibit the Ras downstream signalling pathways in glioblastoma cells. MATERIAL AND METHODS U87 and A172 glioblastoma cells were grown to 80% confluence and treated with Osimertinib in concentrations ranging from 3 μM to 7 μM. After 72 hours cell viability was quantified via XTT-assay. Dose-response curves were calculated by probit analysis. For investigation of the inhibitory effect of OTX008 and Osimertinib on the constitutive activation of Ras U87 cells were treated with 15 μM OTX008, 3 μM Osimertinib, a combination of 15 μM OTX008 and 3 μM Osimertinib and a combination of DMSO and hydrochloricacid. After 72 hours the cells were collected and lysed using Active Ras Detection Kit from Cell Signaling Technology for western blot analysis. RESULTS Osimertinib reduced cell survival in all tested glioblastoma cell lines with a mean ED50 of 6.81 (95%CI: 6.75-6.86) μM. Using western blot analysis to evaluate the effect on the effector protein Ras at the beginning of the PI3K/AKT and RAS/MAPK pathways the effect of OTX008 and Osimertinib on the proliferation of glioblastoma cell line U87 was shown with the inhibition of Ras. CONCLUSION The constitutive activation of the PI3K/Akt and RAS/MAPK pathways is associated with uncontrolled cell growth and therapeutic resistance. The EGFR-inhibitor Osimertinib and the selective Galectin-1 inhibitor OTX008 showed antiproliferative effect in immortalized glioblastoma cell line U87 and were also able to inhibit activation of Ras. Future experiments should focus on the downstream signalling of the pathways, especially in EGFR mutated glioblastoma stem cells.

Comparison of the effect of Sumbawa honey and sucrose as extracellular cryoprotectants on viability, morphology, and phenotype stability of CD34+ hematopoietic stem cells from umbilical cord blood

Cryopreservation is a common practice for the long-term storage and maintenance of the quality of Hematopoietic Stem Cells (HSC) from Umbilical Cord Blood (UCB). Cryomedium containing 10% DMSO is the standard intracellular cryoprotectant agent (CPA) in HSC cryopreservation. However, DMSO is toxic to both cells and patients during transplantation. Therefore, the concentration of DMSO needs to be reduced by adding extracellular CPAs, such as sucrose or Sumbawa honey. The objective of this study was to compare the ability of Sumbawa honey and sucrose as extracellular CPAs to protect HSC CD34+ during cryopreservation. This in vitro study was designed using cryomedium consisting of 10% DMSO as a control, 5% DMSO + 5% Sumbawa honey, and 5% DMSO + 5% sucrose. The results showed that the cryoprotectant containing 5% DMSO + 5% Sumbawa honey had a positive effect and a significant difference (p<0.05) compared with 5% DMSO + 5% sucrose on the viability and morphology of HSC. However, the mean reduction in phenotype stability as indicated by the decrease in percentage CD34+ in the 10% DMSO (6.90 ± 8.60), 5% DMSO + 5% sucrose (10.60 ± 9.20), and 5% DMSO + 5% Sumbawa honey (8.60 ± 11.50) showed no significant difference (p>0.05). In conclusion, the combination of DMSO and Sumbawa honey was able to maintain the viability, morphology, and phenotype stability of HSC. Therefore, honey can be used as an alternative cryoprotectant for the cryopreservation of hematopoietic stem cells.

The Effect of Cryoprotectants Concentration on Ice Crystal Propagation Velocity.

Background: Two physicochemical effects occur during vitrification: nucleation and crystallization. Nucleation is a statistical occurrence by its nature. Thus, the more water molecules that are present the higher are the chances for nucleation to occur. Crystallization is a first-order transition where a water molecule is incorporated into ice crystal. Intracellular viscosity, which is the combination of water, salts, and cryoprotectants (CPs), affects both the nucleation and crystal growth rates. Ice velocity is inversely correlated with the viscosity and directly proportional to the function of the system's supercooling. However, little is known about the speed of ice crystal propagation in vitrification solutions containing different concentrations of CPs. Methods: This article describes the ice crystal propagation velocity while referring to vitrification. Ice crystal propagation velocity was measured in solutions containing different CP (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG], and glycerol) concentrations at a supercooled temperature. The different CPs solutions were inserted into 0.25 mL straws and placed in different temperatures of an alcohol bath (Tc) at supercooling temperatures of -8°C to -10°C. Results: We found that ice crystal propagation is inversely correlated to CP concentrations. Interestingly, PG showed, with statistically significant results, lower ice crystal growth velocities up to concentrations of 30% (v/v), compared with DMSO, and EG at the same concentrations. The combination of EG with PG showed better results (0.25 mm/s) than EG with DMSO (0.39 mm/s) in terms of decreasing the ice crystal growth velocity. When the concentration was increased to 40% (v/v), EG showed the lowest ice crystal propagation velocity (0.09 mm/s), although not significantly different than PG and glycerol but significantly lower than DMSO (0.13 mm/s). Conclusion: These results suggest that current vitrification solutions are not optimized. Based on our results, we suggest that combining PG with EG has advantages over the combination of DMSO and EG, which might promote successful cell and tissue vitrification.

Optimization and application of MTT assay in determining density of suspension cells

MTT assay has been applied widely in stimulation and inhibition tests for adherent cells. However, reading step in regular MTT assays requires medium removal to dissolve formazan by absolute solvents results are usually affected by incomplete formazan dissolution and protein precipitation in diluted solvents. Therefore, most of current MTT assay procedures have difficulties in application on suspension cell lines. In this study, we demonstrated a modified MTT assay in which formazan dissolution method was improved by using the combination of DMSO and SDS-lysis solution. Importantly, the modified MTT assay did not require medium removal, thus it can be applicable for both suspension and adherent cell lines. We also verified that the modified MTT procedure could be effectively applied in bioactivity assays such as cancer cell inhibition and fibroblast stimulation assays. Besides the ease of use, our data regarding nonlinear regression model fitting, data variation, and separation clearly demonstrated that the sensitivity, stability and precision of modified assay were higher than those of common MTT procedures using isopropanol or DMSO as solvents. This study indicated that the modification described here can broaden the MTT assay application on suspension cell lines and also simplify the MTT protocol on adherent cell lines.

Preliminary study: the effect of cryopreservation on the gastrula-staged embryo of African catfish (Clarias gariepinus)

This study was aimed to observe the effect of cryopreservation on gastrula-staged embryo of African catfish. The gastrula-staged embryos were treated 5% (v/v) solutions concentration of dimethyl sulfoxide, propylene glycol, honey, and combined cryoprotectants, respectively and preserved at temperatures of -4 and -196ºC (in liquid nitrogen) for 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. Thawing of embryos was conducted in freshwater at temperature of 28ºC. After thawing, the embryos were incubated in the aquaria at 28ºC temperature. The result showed that the cryopreservation of gastrula-staged embryo at temperatures of -4 and -196ºC affects damage and hatching percentages of African catfish embryos. The percentage of catfish embryo damage increases with the length of preservation at temperature of -4ºC for all treatments. A combination of DMSO+honey and PG+honey has the lowest damage percentage and the highest hatching rate of catfish embryo compared to other treatments (p<0.05). Meanwhile, total embryo damage occurs since the first 30 min of preservation at temperature of -196ºC for all treatments. Cryoprotectant toxicity and inability to protect the embryo are thought to be a cause of damage and death of catfish embryos on preservation, especially at temperature of -196ºC.

Quality assessment of cryopreserved New Zealand white rabbit spermatozoa in INRA-82 extender containing different cryoprotectants

&lt;p&gt;The present study aimed to evaluate the effect of supplementation of INRA-82 semen extender with different cryoprotectants (dimethyl sulphoxide; DMSO vs. dimethyl formamide; DMF) on the quality of white New Zealand rabbit buck spermatozoa. We also investigated the possible association between the synergistic action of DMSO and DMF and their relation with INRA-82 extender composition. Semen was collected and pooled from 8 adult rabbit bucks. Pooled semen samples were diluted 1:1 with INRA-82 extender supplemented with DMSO 8%, DMF 8% or a combination of DMSO 4% and DMF 4%. The diluted semen samples were cryopreserved in 0.25 plastic straws. After thawing, progressive motility, sperm viability, sperm abnormalities, membrane integrity, acrosome status, viability index and DNA integrity were evaluated. The results showed that dilution of rabbit buck semen in INRA-82 supplemented with DMSO and DMF (4% each) before freezing significantly (&lt;em&gt;P&lt;/em&gt;&amp;lt;0.05) improved sperm motility (42.00%), percentage of live spermatozoa (45.30%), proportions of spermatozoa with intact acrosome (59.75%) and percentage of spermatozoa with non-fragmented DNA (86.04%), compared to those diluted in INRA-82 supplemented either with DMSO 8% (+9, +10, +5 and +7 percentage points, respectively) or with DMF 8% alone (+18 +18, +12 and +9 percentage points, respectively). In conclusion, dilution of rabbit buck semen before freezing with INRA-82 extender supplemented with a combination of DMSO 4% and DMF 4% improved quality of frozen-thawed New Zealand White rabbit spermatozoa. Furthermore, our results also suggest that supplementation of INRA-82 with DMSO or with DMF alone at higher concentrations deteriorates the sperm quality.&lt;/p&gt;

A Screening of Permeation Enhancers for Transdermal Delivery of Propofol

Propofol is a highly lipophilic anesthetic drug used to induce sedation, predictable amnesia and anxiolysis. In this project, several permeation enhancers were screened to improve the permeation of propofol across the porcine full thickness skin. The screening studies of permeation enhancers was performed in two phases. As a result of the first phase of screening studies, DMSO, Laureth-4 and transcutol were found to be the potential penetration enhancers which improved the permeation of propofol across the skin ~ 4, 3.5 and 4.5-fold more compared to control. A 100% propofol was used as a control. In the second phase, the combination of DMSO and Laureth-L4 (F1) delivered 278.6 ± 20.3 µg/cm2 propofol across the skin which were found to be ~ 18 more compared to control (15.68 ± 2.6 µg/cm2 ). The studies in this project concluded that the combination of DMSO and Laureth-L4 can be used for the development of transdermal propofol product in the future.

Criopreservação do sêmen do Prochilodus lineatus: efeito da combinação de crioprotetores

The study was conducted with the objective of comparing the toxicity and the effect of the combination of intra and extracellular cryoprotectants in curimba Prochilodus lineatus sperm cells subjected to cryopreservation. Semen from 19 males were analyzed and diluted in four solutions comprise of intra and extracellular cryoprotectants in the following combinations: methanol+lactose, methanol+egg yolk, DMSO+lactose and DMSO+egg yolk. A portion of the diluted semen was frozen while the remaining fraction was kept in repose and evaluated after 10 min. For freezing, the diluted samples were packaged in 0.50 mL straws and placed into liquid nitrogen vapor for 24 h and, after this time, submerged into liquid nitrogen for 10 days. The combination of DMSO+lactose was less toxic to the diluted semen, resulting in higher motility rates and durations when compared to the other treatments. After thawing, the highest motility rate and duration were obtained using lactose as extracellular cryoprotectant, regardless of its combination. There was no significant difference between treatments when analyzing sperm morphology after thawing. Considering the effects of the tested treatments, the use of lactose as an extracellular cryoprotectant added with DMSO or methanol is the most suitable, since these combinations presented the highest motility rates and durations and low morphological change rate after thawing.

Comparative study of the physical, chemical, and multimodal approaches to enhancing nanoparticle transport in the skin with model dermatitis

This paper presents a comparative analysis of the combined and separate influence of ultrasound and DMSO on the transport of a gold nanoshell suspension in intact and injured skin from data on optical coherence tomography and histochemical analysis. Experimental allergic contact dermatitis was used to model injury to the stratum corneum during various pathological changes in the skin. The studies were performed on outbred laboratory rats. It is shown that the best method for enhancing transdermal transport of an immersion liquid is multimodal physical and chemical impact (a combination of DMSO and ultrasonophoresis); the effectiveness of optical clearing of the dermis both in the presence and absence of the stratum corneum is approximately the same. To enhance the transport of nanoparticles into the skin when it undergoes pathological changes related to injuries of the protective barrier, exposure to ultrasound is sufficient.

Pd‐catalyzed ligand‐free desulfitative Heck reaction with arenesulfinic acid salts under air

Palladium‐catalyzed cross‐coupling reactions of various aryl sulfinic acid salts with a wide variety of vinyl substrates have been achieved in good to excellent yields under simple aerobic conditions at 70°C with the assistance of Cu(II) salts. The reaction can be accelerated by the combination of DMSO with THF. The reported Matsuda–Heck type coupling reactions are tolerant to the common functional groups, making these transformations as attractive alternatives to the traditional cross‐coupling approaches. Copyright © 2013 John Wiley &amp; Sons, Ltd.

ChemInform Abstract: Active Thionium Species Mediated Functionalization at the 2α‐Position of Indole Derivatives.

AbstractThe 2α‐functionalization of indole derivatives such as tetrahydrocarbazoles and the pyridoindole derivative (VII), a core structure of biologically active natural products, is achieved via activation through a combination of DMSO and TFAA.

A Novel Approach to Embryo Vitrification by LN 2 Vapor Storage of Stripper Tips in CBS High-Security Straws

Introduction: Embryo vitrification first succeeded in 1986 (Rall and Fahey). Since then various vitrification solutions (VS) and container types have been investigated. The combination of DMSO and EG (≥30% v/v) has been widely applied as the VS of choice. Owing to the sensitive nature of osmotic stress (“toxicity”) in high-molar VS, the ease of technique application and technician consistency are important factors. Additionally, there is increasing concern over pathogen cross-contamination and the safe long-term storage of gametes and embryos in LN2. Objective: To develop a vitrification procedure using FDA-approved devices/solutions that is effective, easily repeatable, and offers secure contamination-free LN2 storage, before we implement vitrification into our clinical egg and embryo cryopreservation plan. Methods: Frozen 1-cell mouse embryos (Embryo-Tech) were cultured to expanded/hatching blastocyst (BL) in tri-gas Sanyo mini-incubators for proficiency testing purposes. Fifty good-quality BL were randomly assigned to either a control group (Grp 1) or a vitrification treatment (Grp 2). In Grp 1, BL were diluted in 1.5 mol/L glycerol solution for 10 min + 0.2 mol/L sucrose for 5 min before being loaded and sealed in CBS straws and slow cooled from −7°C to −35°C at 0.5°C/min. Embryos were thawed using a standard Irvine Scientific BL Thaw kit. In Grp 2, BL were vitrified and thawed using the Irvine Scientific VS Cryo and Thaw kits as described, the primary difference being that the 275-μm denuding pipette (Stripper Tip) used for embryo handling was also used as the final VS-embryo storage container (approx. 2 μL volume). Each Stripper Tip was shortened about 1.5 cm to accommodate placement into a prelabeled/sealed CBS straw, tip-end first, and then sealed again (both sides securely sealed) using a Symm sealing device before direct placement into LN2 (plunged within 1 min of final VS exposure). Each straw contained 5 BL/straw. Results: No difference was observed between treatment groups, with 17 of 25 BL (68%) in both Grp 1 and Grp 2 re-expanding and continuing development 24 h after thaw. Conclusions: A recent report by Isachenko et al. (Cryobiol 54:305) indicated that a double-straw container system can reduce the ultra-rapid cooling rate of vitrification up to 25-fold (15,000°C/min in open container versus 600°C/min, respectively) without any effect on BL survival. In this study, the use of CBS straws to contain vitrified embryos within a denuding pipette has proven to be a simple and effective approach to contamination-free cryostorage.

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0 h after T 0). The K4 cooling programme was used to freeze 1 ml of cryoextended sperm using 1.8 ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5 ml straws and compared these with 1.8 ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10 s post-activation. Sperm was collected directly into IS and stored at 4 °C for 2.5 h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8 ml cryotubes or straws (0.5, 1 and 5 ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to −9 °C at a rate of 4 °C min −1 and then from −9 to −80 °C at a rate of 11 °C min −1) and a rapid cooling programme (b) named L1 (directly from +4 to −80 °C at a rate of 20 °C min −1). Both slow (K4) and rapid (L1) cooled samples were held 6 min at −80 °C. Finally, samples were transferred into liquid N 2. The frozen spermatozoa were thawed in a water bath (40 °C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0 h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5 ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46 μm s −1 and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

‘Reductive ozonolysis’ via a new fragmentation of carbonyl oxides

This account describes the development of methodologies for ‘reductive’ ozonolysis, the direct ozonolytic conversion of alkenes into carbonyl groups without the intermediacy of 1,2,4-trioxolanes (ozonides). Ozonolysis of alkenes in the presence of DMSO produces a mixture of aldehyde and ozonide. The combination of DMSO and Et 3N results in improved yields of carbonyls but still leaves unacceptable levels of residual ozonides; similar results are obtained using secondary or tertiary amines in the absence of DMSO. The influence of amines is believed to result from conversion to the corresponding N-oxides; ozonolysis in the presence of amine N-oxides efficiently suppresses ozonide formation, generating high yields of aldehydes. The reactions with amine oxides are hypothesized to involve an unprecedented trapping of carbonyl oxides to generate a zwitterionic adduct, which fragments to produce the desired carbonyl group, an amine, and 1O 2.

Cryopreservation of adherent neuronal networks

Neuronal networks have been widely used for neurophysiology, drug discovery and toxicity testing. An essential prerequisite for future widespread application of neuronal networks is the development of efficient cryopreservation protocols to facilitate their storage and transportation. Here is the first report on cryopreservation of mammalian adherent neuronal networks. Dissociated spinal cord cells were attached to a poly- d-lysine/laminin surface and allowed to form neuronal networks. Adherent neuronal networks were embedded in a thin film of collagen gel and loaded with trehalose prior to transfer to a freezing medium containing DMSO, FBS and culture medium. This was followed by a slow rate of cooling to −80 °C for 24 h and then storage for up to 2 months in liquid nitrogen at −196 °C. The three components: DMSO, collagen gel entrapment and trehalose loading combined provided the highest post-thaw viability, relative to individual or two component protocols. The post-thaw cells with this protocol demonstrated similar neuronal and astrocytic markers and morphological structure as those detected in unfrozen cells. Fluorescent dye FM1-43 staining revealed active recycling of synaptic vesicles upon depolarizing stimulation in the post-thaw neuronal networks. These results suggest that a combination of DMSO, collagen gel entrapment and trehalose loading can significantly improve conventional slow-cooling methods in cryopreservation of adherent neuronal networks.

Effect of Different Cryoprotectants on the Survivability and the Development of Bovine Oocytes Matured In vitro

Tsuzuki. Y., Kusao. T., Ashizawa, K. and Fujihara, N. 2000. Effect of different cryoprotectants on the survivability and the development of bovine oocytes matured in vitro. J. Appl. Anim. Res., 18: 15–24. To determine the optimum combination of cryoprotectant and freezing method, for improving the developmental, competence of in vitro matured bovine oocytes, three cryoprotectants (1.5 M dimethyl-sulfoxide + 0.25 M sucrose: DMSO; 0.9 M ethylene glycol + 0.8 M propylene glycol + 0.1 M sucrose: EG + PG; 0.8 M PG + 0.8 M DMSO: PG + DMSO) and two freezing methods [the oocytes were cooled from 0C to −6.5C at 1 C/min, seeded and cooled to −30C at 0.3C/min (method A); the oocytes were cooled at −8C directly, seeded and cooled to −35C at 1 C/min (method B)] were used. The survivability and developmental competence of the oocytes thawed from the liquid nitrogen were determined by the dye extrusion assay with eosin and embryonic development after in vitro fertilization, respectively. Without freezing, the survivability of the oocytes equilibrated with and removed from each cryoprotectant were not decreased compared with that in the control group. However, the per cent of the oocyte developed to the blastocyst stage in all of the cryoprotectant treated groups were significantly lower (P<0.05) than that of the control group. DMSO group frozen with method A and EG + PG group frozen with method B maintained their survivability compared to the each control group. However, cleavage rates in all groups were significantly lower (P<0.05)than those of the control groups. No blastocyst was observed in all frozen groups. These results suggest that the combination of DMSO with freezing method A and EG + PG with freezing method B seemed to be more suitable compared with the other combinations.

Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content.

A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaq1 and Pfu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.

Preliminary Study on Cryopreservation of the Conchocelis of Porphyra yezoensis.

Cryopreservation of the conchocelis of Porphyra yezoensis was attempted. Conchocelis cells were frozen at -30 or -80°C in various concentrations of seawater containing several cryopro-tectants. The viability of conchocelis cells was evaluated by staining them with neutral red. DMSO was the best cryoprotectant among the compounds tested. The cryoprotective effect of DMSO varied with the concentration of seawater, and was maximal at a seawater concentration of 50%. The combination of DMSO and sorbitol was more effective for the long survival of conchocelis than DMSO alone. The survival rate of conchocelis frozen at -80°C in 50% seawater with 5% DMSO and 0.5M sorbitol fell below 1% in three days. However, when frozen at -30°C, 55% of the conchocelis cells survived for one day and 16% survived for a week.