DMSO Exposure Research Articles

TPD-seq: A high throughput RNA-seq method to derive transcriptomic points of departure from cell lines.

There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput in vitro experiments. To further help democratize tPOD research, here we outline 'TPD-seq' which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). We applied the TPD-seq workflow to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H2O2) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (~9 k for fish cells; ~6 k for human cells), and about 50 % of differentially expressed genes were curve-fitted from which 90 % yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31-155 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ~5 to 76 μM. Finally, exposure of the two human cell lines to H2O2 resulted in tPOD values that ranged from 0.7 to 1.1 mM in Caco-2 cells and 5-30 μM in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the TPD-seq workflow can be determined with additional case studies.

The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14–49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.

SAT427 Effects Of Polychlorinated Biphenyls (PCBs) On Transcription Factor NFκB Activation In Neonatal Rat Microglia Primary Culture

Abstract Disclosure: C.E. Dressel: None. G.M. Valdez: None. M.R. Bell: None. Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are lipophilic and accumulate in the food chain. Despite bans on PCB production, human exposure continues and can cause disruptions in endocrine, nervous, and immune system functions. As such, PCBs may interfere with the actions of microglia, the innate immune cells of the central nervous system. Healthy microglia are critical for neural development, maintenance of neural functions, and responses to stress, injury, or infection. Initial studies of primary neonatal rat microglial cultures indicate that PCBs alter gene expression of proinflammatory cytokine TNF-α and IL-1β in response to a secondary inflammatory lipopolysaccharide (LPS) challenge. Production of these cytokines is, in part, regulated by transcription factor NFκB. This experiment tests the hypothesis that PCBs alter NFκB activation in neonatal microglia. The forebrains of male and female neonatal Sprague-Dawley rats were dissected to remove meninges, homogenized with collagenase and gentle trituration, and plated on coverslips in DMEM FBS+ media to establish a primary mixed glial culture. After one week, microglia were subcultured using mild trypsin treatment and maintained in conditioned media for 24 hours. The microglia were then exposed to either PCB 153 (0.1 µM) or DMSO vehicle control (0.036%) for 18 hours. After 18 hours, the cells were treated with either LPS (0.5 µg/mL) or PBS vehicle control for 4 hours and then fixed. Immunocytochemistry was used to visualize NFκB and identify microglia, with anti-p65 and Ox42 antibodies, respectively. Coverslips were mounted with DAPI-containing media and the cells were imaged via confocal microscopy. In response to immune activation, NFκB typically translocates from the cytosol to the nucleus. Therefore, we assessed NFκB activation by quantifying p65 fluorescent pixel intensity and percentage of area covered in nuclear or cytoplasmic regions according to DAPI and Ox42 channel masks using ImageJ. A two-way ANOVA was used to identify the effects of PCB vs. DMSO exposure and LPS vs. PBS challenge. Preliminary data indicated that LPS increased the ratio of nuclear to cytosolic NFκB, as expected. PCB exposed cells tended to have lower NFκB presence in the nucleus and cell as a whole, but a greater proportion of NFκB staining localized within the nucleus relative to the rest of the cell. With the current sample (n=4-6 per group, 2-3 males and females each), no interactions between PCBs and LPS were observed. Continued work will increase sample size to explore sex differences and the time course of NFκB response to PCBs, which may help broaden our understanding of the mechanisms by which PCBs affect normal brain activity and disrupt healthy endocrine, immune, and neural development. Presentation: Saturday, June 17, 2023

Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 μM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 μM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.

Cryopreservation OfPyramimonas Mucifera

BACKGROUND:It is important to appreciate microalgal diversity, better understand their ecosystem functioning and therefore implement conservation measures. The National Biodiversity Act of South Africa has a marine and coastal component which promotes such investigations.OBJECTIVE:To develop a cryostorage method for the marine unicellular algal speciesPyramimonas mucifera.MATERIALS AND METHODS:Cell viability, measured by propidium iodide, was used to determine both optimal exposure time to 10 % DMSO and survival following thawing of cryopreserved cells. Cryopreservation was achieved by a two-step cooling method.RESULTS & DISCUSSION:A 30-min DMSO exposure was selected forP. mucifera, as cells following such treatment retained cell shape and integrity. Although density was significantly reduced after cryopreservation, the surviving cells were capable of returning to viability levels equal to those of the untreated control (>90%).CONCLUSION:Cultures ofP. muciferacan be successfully cryopreserved and propidium iodide provides a useful indication of culture vitality.

The Solvent Dimethyl Sulfoxide Affects Physiology, Transcriptome and Secondary Metabolism of Aspergillus flavus.

Dimethyl sulfoxide (DSMO) is a simple molecule widely used because of its great solvating ability, but this solvent also has little-known biological effects, especially on fungi. Aspergillus flavus is a notorious pathogenic fungus which may contaminate a large variety of crops worldwide by producing aflatoxins, endangering at the same time food safety and international trade. The aim of this study was to characterize the effect of DMSO on A. flavus including developmental parameters such as germination and sporulation, as well as its transcriptome profile using high-throughput RNA-sequencing assay and its impact on secondary metabolism (SM). After DMSO exposure, A. flavus displayed depigmented conidia in a dose-dependent manner. The four-day exposition of cultures to two doses of DMSO, chosen on the basis of depigmentation intensity (35 mM “low” and 282 mM “high”), led to no significant impact on fungal growth, germination or sporulation. However, transcriptomic data analysis showed that 4891 genes were differentially regulated in response to DMSO (46% of studied transcripts). A total of 4650 genes were specifically regulated in response to the highest dose of DMSO, while only 19 genes were modulated upon exposure to the lowest dose. Secondary metabolites clusters genes were widely affected by the DMSO, with 91% of clusters impacted at the highest dose. Among these, aflatoxins, cyclopiazonic acid and ustiloxin B clusters were totally under-expressed. The genes belonging to the AFB1 cluster were the most negatively modulated ones, the two doses leading to 63% and 100% inhibition of the AFB1 production, respectively. The SM analysis also showed the disappearance of ustiloxin B and a 10-fold reduction of cyclopiazonic acid level when A. flavus was treated by the higher DMSO dose. In conclusion, the present study showed that DMSO impacted widely A. flavus’ transcriptome, including secondary metabolism gene clusters with the aflatoxins at the head of down-regulated ones. The solvent also inhibits conidial pigmentation, which could illustrate common regulatory mechanisms between aflatoxins and fungal pigment pathways. Because of its effect on major metabolites synthesis, DMSO should not be used as solvent especially in studies testing anti-aflatoxinogenic compounds.

Exposure to DMSO during infancy alters neurochemistry, social interactions, and brain morphology in long-evans rats.

IntroductionDimethyl sulfoxide (DMSO) is a widely used solvent to dissolve hydrophobic substances for clinical uses and experimental in vivo purposes. While usually regarded safe, our prior studies suggest changes to behavior following DMSO exposure. We therefore evaluated the effects of a five‐day, short‐term exposure to DMSO on postnatal infant rats (P6‐10).MethodsDMSO was intraperitoneally injected for five days at 0.2, 2.0, and 4.0 ml/kg body mass. One cohort of animals was sacrificed 24 hr after DMSO exposure to analyze the neurometabolic changes in four brain regions (cortex, hippocampus, basal ganglia, and cerebellum) by hydrophilic interaction liquid chromatography. A second cohort of animals was used to analyze chronic alterations to behavior and pathological changes to glia and neuronal cells later in life (P21‐P40).Results164 metabolites, including key regulatory molecules (retinoic acid, orotic acid, adrenic acid, and hypotaurine), were found significantly altered by DMSO exposure in at least one of the brain regions at P11 (p < .05). Behavioral tests showed significant hypoactive behavior and decreased social habits to the 2.0 and 4.0 ml DMSO/kg groups (p < .01). Significant increases in number of microglia and astrocytes at P40 were observed in the 4.0 ml DMSO/kg group (at p < .015.)ConclusionsDespite short‐term exposure at low, putatively nontoxic concentrations, DMSO led to changes in behavior and social preferences, chronic alterations in glial cells, and changes in essential regulatory brain metabolites. The chronic neurological effects of DMSO exposure reported here raise concerns about its neurotoxicity and consequent safety in human medical applications and clinical trials.

The association between oxidative stress-induced galectins and differentiation of human promyelocytic HL-60 cells

Galectins are multifunctional β-galactoside-binding proteins that are involved in the regulation of cellular stress responses and differentiation. The relationship between these processes is unclear and we report here that galectins display oxidative-stress specific expression patterns in neutrophil-like differentiated HL-60 cells. Three galectins (−1, −3, and −10) are upregulated in response to either menadione or DMSO exposure whereas galectins −9 and −12 exhibited a stimulus-dependent downregulation. Changes in galectin expression are oxidant dependent based on the observations that 1) oxidative stress biomarkers HMOX1 (heme oxygenase-1) and NCF1 (neutrophil cytosolic factor 1, which is also a biomarker of neutrophil differentiation) are elevated in both cases, and 2) the antioxidant N-acetyl-L-cysteine restores basal expression of galectin-3 following oxidant exposure. In addition, our results suggest that the regulation of oxidative stress-sensitive galectins involves DNA hypomethylation mechanisms. Expression of galectin-3 and galectin-12 exhibits an opposite relationship to the expression of HMOX1/NCF1, suggesting a stimulatory and inhibitory role of these galectins in neutrophil-like differentiation of HL-60 cells. We also show that the inhibition of galectins reduces the growth rate of HL-60 cells, and facilitates their neutrophil-like differentiation. Collectively, our findings indicate that the process of cellular differentiation implicates, in part, oxidative stress-sensitive galectins, which further highlights a biological significance of galectin network remodeling in cells.

S100A4 (metastasin) positive mesenchymal canine mammary tumour spheroids reduce Tenascin C synthesis under DMSO exposure in vitro.

In breast cancer research S100A4-positive tumour-associated stromal cells are assumed as primary source of Tenascin C (TNC) in the metastatic environment. Aim of the present study was to isolate and characterize S100A4/TNC positive stromal canine mammary tumour (CMT) cells. Cells grown as scaffold-free spheroids were investigated for S100A4, TNC, and proliferative activity under 1.8% DMSO stimulation by means of Western blot and immunohistochemistry. DMSO is a commonly used drug solvent despite well-known side effects on cells including TNC expression. DMSO did not affect proliferation, but TNC was significantly reduced under DMSO exposure for 7 and 14 days, whereby for S100A4 a reducing effect was only observed after 14 days. Without DMSO, cells stably expressed TNC and S100A4 which makes them suitable to be used in experimental approaches requiring S100A4/TNC expressing CMT stromal cells. Results show that 1.8% DMSO should not be used as solvent for experiments concerning TNC/S100A4 expression.

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality.

LC-MS/MS determination of tralopyril in water samples.

A targeted analytical method was established to determine tralopyril (4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile) in water. This compound has been recently introduced as a biocide in ship antifouling paints, becoming a potential new environmental contaminant. The method presented here allows for the first time the direct determination of tralopyril in environmental samples without the need of a pre-concentration step. The injected sample is separated by a 30 min HPLC-gradient on a reversed phase column and the compound identified and quantified by negative ion LC-MS/MS. Tralopyril solutions in DMSO, seawater, river Glatt water and E3 medium (used for zebrafish experiments) were analysed to demonstrate the applicability of the method. The method provides good retention time reproducibility and a quantitation limit (LOQ) of 0.025 μg L(-1) for DMSO, seawater and E3 exposure medium and 0.05 μg L(-1) for river Glatt water. Calculated tralopyril half-lives were 6.1 h for seawater, 8.1 h for river Glatt water and 7.4 h for E3 medium at 18 °C.

BCL-Xl Contributes to Decreased Cellular Respiration During Erythroid Differentiation

AbstractAbstract 3201The BCL2 family of proteins are well known for their ability to both positively and negatively regulate mitochondrial mechanisms of apoptosis. The anti-apoptotic members of this family can decrease mitochondrial outer membrane permeability and cytochrome c release. By stabilizing the cell against apoptosis, these proteins allow cell survival even in states of low energy. However, despite this intimate link between the BCL2 family proteins and mitochondria, their direct effect on metabolism is less clearly understood. It is generally expected that metabolic changes induced by the BCL2 family of proteins will further impact cell survival as murine hepatoma and cancer cells overexpressing Bcl-xL are sensitive to Bcl-xL inhibition1 but BCL-xL is also known to be essential for erythroid differentiation and more recently was linked specifically to heme synthesis2. We therefore set out to investigate whether there was a connection between BCL-xL induced changes in cellular respiration and erythroid differentiation.Murine erythroleukemia (MEL) cells were differentiated by exposure to 2% DMSO for 5 days and then real time oxygen consumption was measured on the Seahorse extracellular flux analyzer (XFA). DMSO induced differentiation yielded a 4-fold decrease in oxygen consumption. Western blot analysis revealed that BCL-xL was induced during differentiation. We then generated cell lines in which BCL-xL was knocked down with small hairpin RNA (shRNA). As differentiation has previously been reported to be fatal in MEL cells without BCL-xL activity, both parental cells and BCL-xL knockdowns were infected with a vector over expressing BCL2. Differentiation over 5 days with 2% DMSO was performed on these new cell lines. Erythroid differentiation was confirmed using Benzidine staining. While the control cell line showed high rates of Benzidine staining after exposure to DMSO, the BCL-xL knockdown cell line consistently showed <5% benzidine positivity. Western blot analysis confirmed the absence of BCL-xL induction by DMSO exposure in the knockdown cell line. Using the Seahorse XFA the control cell line was shown to have significant decrease in oxygen consumption when exposed to DMSO, while DMSO exposed BCL-xL knockdown cells showed less than half this drop in oxygen consumption. However, both control and BCL-xL knockdowns have limited respiratory reserve as the response to CCCP, an uncoupler of electron transport, is diminished after DMSO exposure as compared to their undifferentiated counterparts.Our results suggest that erythroid differentiation is associated with a significant decrease in cellular respiration. Although, not the only contributor to the decreased dependence on oxidative phosphorylation of cells undergoing erythroid differentiation, BCL-xL expression is clearly a necessary factor. Our data is able to connect BCL-xL expression to both erythroid differentiation and this distinct metabolic phenotype. As BCL-xL's role in erythroid differentiation has previously been reported to be associated with heme synthesis, future work will focus on identifying oxidative metabolic pathways associated with BCL-xL expression and heme synthesis. Disclosures:No relevant conflicts of interest to declare.

DMSO Reduction Strategies Reduce DMSO Induced Post Autologous Transplant Morbidity In Patients with Lymphoma and Myeloma –Results from an EBMT Non Interventional Prospective Study

AbstractAbstract 2397 Introduction.DMSO is the universally used cryoprotectant for freezing peripheral blood stem cell for autologous transplantation. DMSO has a significant side effect profile including vasoconstriction, nausea and vomiting and with transplant related mortality now in the region of 1–3% avoiding DMSO toxicity is of increasing importance. Methods.The study was questionnaire based looking at centres freezing strategy(s), administration of stem cells to patients and individual forms for each patient transplanted with follow up form for side effect analysis. Side effects were defined using NCI criteria (version 3.0) and assigned to DMSO (or not) by the reporting centres. On account of substantial centre variation noted at initial analysis results were analysed using the Mantel-Haenszel Odds Ratio (OR) averaged over the centres. Results.64 centres contributed data for a maximum of 12 months between January 2007 and December 2008. A total of 1651 patients, 1008 male and 637 female, age range 18–76, median 56, with 875 myeloma patients, 540 NHL, 220 Hodgkin's Disease and 16 CLL were used for analysis. Almost all myeloma and most of the other patients had melphalan based regimens. From the Centre survey it was noted that 51 centres used 10% DMSO (1273 patients) but on account of multiple centre strategies 15 centres used concentrations down to 5%. Three Centres using 10% DMSO washed cells before infusion while another 9 Centres washed cells occasionally (>10% of transplants). The median amount of DMSO given per patient was 20ml although the upper limit per Centre was often very much higher. 75% of Centres did not use any delay between bags of stem cells and the median duration of infusion was 22 minutes.440 patients experienced nausea or vomiting related to DMSO, 123 hypo- or hypertension and 84 other side effects. The side effect profile was then amalgamated so that no patient was counted more than once. DMSO was calculated in ml/Kg body weight for each patient then split into quartiles for analysis. The OR for any side effect assigned to DMSO between patients in the highest quartile and patients in the lower three quartiles averaged over age and disease classification was 1.7 (Confidence Interval 1.2 to 2.4, p = 0.003). When split into groups by disease (lymphoma or myeloma) and age (>median and <median) the OR were as follows: young lymphoma 1.6 (0.95 to 2.8) older lymphoma 3.6 (1.5 to 9.1) younger myeloma 0.8 (0.34 to 2.0) older myeloma 1.8 (0.95 to 3.5). The reason for the apparent protective effect in younger myeloma is subject to further analysis but we suggest that in this patient group in particular the effect of the DMSO is obscured by the side effects of the melphalan; this hypothesis is supported by an analysis of all side effects. When results were calculated as DMSO per ml/min it appeared possible that the quicker the DMSO is given the less the adverse effects. Conclusions.DMSO contributes to the morbidity of patients undergoing autologous transplantation. Strategies to reduce DMSO exposure can reduce these complications. Disclosures:No relevant conflicts of interest to declare.

Significantly Lower Cryopreservation Volumes for PBSC Collections Using Spectra Optia® Version 5 Software Compared with the MNC Setting on COBE® Spectra, Particularly When Using Plerixafor for Mobilisation

AbstractAbstract 825Plerixafor (Mozobil®, Genzyme), an antagonist of the alpha chemokine receptor CXCR4, is highly effective as a novel PBSC mobilisation agent for patients failing to mobilise PBSC by conventional means, and is now licensed for this indication in Europe and the USA. However, significantly larger cryopreservation volumes have been reported as a disadvantage when plerixafor is used as part of PBSC mobilisation regimes (Tanhehco et al., Journal of Clinical Apheresis 2010, advance online publication). Although not an insurmountable problem in our own experience, this does increase liquid nitrogen freezer space requirements and also increases DMSO exposure for the patient at the time of re-infusion. In the course of a pre-marketing audit of the Version 5 PBSC collection software for the new Spectra Optia® apheresis platform (CaridianBCT), we observed significantly lower cryopreservation volumes for plerixafor collections when using Spectra Optia® than when using the widely-used mononuclear cell (MNC) setting (“Version 4.1”) on the conventional COBE® Spectra (Caridian BCT) apheresis platform. Formal audit was carried out of a series of 21 consecutive aphereses for PBSC procurement from 16 patients where plerixafor had been used as part of the conditioning regime, with Spectra Optia® being used for 10 procedures and COBE® Spectra for 11. Cryopreservation volume was strikingly and significantly lower for the Spectra Optia® procedures (median 351 mls; range 170 – 716 mls) than for COBE® Spectra procedures (median 756 mls; range 524 – 2400 mls; p<0.001). Extension of this audit to a comparator group of 26 procedures on a temporally matched series of 18 consecutive patients where plerixafor was not used as part of the conditioning regime, of which Spectra Optia® was used for 7 procedures and COBE® Spectra for the remainder, did show lower cryopreservation volume for non-plerixafor collections compared with the 22 plerixafor collections though this did not reach statistical significance (p=0.11). The total white cell count was noted to be significantly higher on the day of PBSC collection for the plerixafor group (median WCC 29.8 × 10⋀9/l) compared to the non-plerixafor group (median WCC 15.1 × 10⋀9/l; p<0.001). Slightly lower cryopreservation volumes were seen in the non-plerixafor group when comparing the Spectra Optia® procedures to the COBE® Spectra procedures, but this was not statistically significant (median for Optia 354 mls, range 99–668 mls compared with median for Spectra 488 mls; range 298–954 mls; p>0.05). However, comparison of Spectra Optia® collections with COBE® Spectra collections for all 47 procedures (plerixafor plus non-plerixafor collections) showed significantly lower cryopreservation volumes for Spectra Optia® (p<0.01). Initial results with Spectra Optia® have shown that it yields a purer product than COBE® Spectra MNC, with lower granulocyte contamination, while maintaining the high PBSC collection efficiency of COBE® Spectra MNC (CaridianBCT, personal communication). In our experience, this translates into a greater than 50% reduction in median cryopreservation volume in the context of collection procedures where plerixafor is used as part of the conditioning regime, probably because of the higher total peripheral WCC seen in plerixafor-mobilised patients which leads to significant granulocyte contamination of the product with older cell separator platforms. Spectra Optia® therefore shows great promise as a solution to the problem of higher cryopreservation volumes when using plerixafor. Disclosures:Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sinclair:Caridian BCT: Honoraria. Douglas:Genzyme Corporation: Consultancy; Caridian BCT: Honoraria.

Characterization of cell‐banking parameters for the cryopreservation of mammalian cell lines in 100‐mL cryobags

This article describes a cell banking process for rBHK cell lines in 100-mL cryobags. As the use of larger volume cell banks requires greater cell numbers and longer preparation time, extensive characterization of key process parameters beyond the conventional ranges was performed to support a cGMP banking process. All experiments were conducted using two recombinant BHK21 cell lines, one of them cotransfected with Hsp70. The results show that the entire cell banking process for these BHK cell lines can be performed at room temperature. A DMSO exposure time up to 5 h either directly in a bioreactor or in shaker flasks did not result in any significant negative effect after cell thaw, when the cryocontainers were frozen immediately after filling. Extensive characterization did not indicate any significant apoptotic effects after thaw. However, the Hsp70 cotransfected cell line did show a slightly better protection from potential cryopreservation-induced apoptosis. Surprisingly, it was found that cells transferred into cryobags showed a low recovery rate after thaw if the incubation time exceeded 1.5 h before freezing. Additional experiments confirmed that the DMSO exposure time inside the cryocontainer in contrast to the DMSO exposure in a reactor or shaker flasks is much more critical. The cryobag cell banking process should therefore be performed within a 1(1/2)-2 h window; a banking process for vials should not exceed 2(1/2) h.

155. Effect of methanol and DMSO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish Danio rerio

155. Effect of methanol and DMSO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish Danio rerio

Extraction, Identification, and Functional Characterization of a Bioactive Substance From Automated Compound-Handling Plastic Tips

Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts.

Expression of DLK/PREF1 Results in Inhibition of Human Myeloid Cell Differentiation and Proliferation through Distinct Molecular Mechanisms.

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. Microarray based analyses have shown that the Delta-Like/Preadipocyte-Factor 1 (DLK/PREF1) gene is overexpressed in CD34+ cells from patients with MDS. DLK encodes an EGF-like transmembrane protein subject to proteolytic cleavage and release. Stromal expression of DLK appears to play a role in maintenance of murine hematopoietic stem cells and in supporting B- and T-lymphopoiesis. However, the functional consequences of DLK overexpression in human hematopoietic cells are not known. We have studied the effects of ectopic expression of DLK on the differentiation and growth of HL-60 promyelocytic cells, and found that ectopic expression of DLK inhibits induced differentiation in response to ATRA, PMA and DMSO exposure. DLK mutants were generated to explore mechanisms underlying its effects on hematopoietic cells. These included DLK-EC, expressing only the extracellular portion of the molecule; DLK-dIC, a transmembrane form lacking the intracellular domain; and DLK-ALT, an alternatively spliced form lacking the protease sensitive site. We observed that proteolytic cleavage and release of the extracellular domain was not required for inhibition of hematopoietic cell differentiation, whereas intracellular domain interactions were critical to this DLK function. We have also tested the effect of DLK expression on myeloid cell proliferation. DLK expressing cells demonstrated significantly reduced proliferation compared with controls (91-fold expansion of control cells after 7 days culture vs. 66-fold expansion for DLK expressing cells, p<0.0001). Studies using SNARF-1 labeling to track cell division confirmed the inhibitory effect of DLK expression on proliferation (proliferation index of 6.39±1.0 for control vs. 4.99±0.3 for DLK expressing cells, p=0.04, n=3). Cell cycle analysis revealed that DLK expressing cells exhibited slower progress through G0/G1 phase into S-phase than controls following release from G2/M phase synchronization with Nocodazole treatment (45±5% of DLK expressing cells vs. 66±10% of control cells were in S-phase after 14 hours, p=0.027, n=3). We also evaluated the effects of mutations in the DLK molecule on its anti-proliferative effect. DLK-dIC inhibited cell proliferation to a similar extent as wild type DLK (p=0.42), whereas the DLK-ALT resulted in further reduction in proliferation (p=0.0014). In contrast, the DLK-EC resulted in significantly enhanced cell proliferation compared with wild type DLK (p<0.0001). These results indicate that the inhibitory effects of DLK on proliferation require its expression as a transmembrane molecule but do not require intracellular domain mediated interactions. On the other hand proteolytic cleavage and release of the extracellular domain appears to have a stimulatory effect on cell proliferation. In summary, DLK overexpression in myeloid cells has important functional consequences including inhibition of proliferation and differentiation. The inhibitory effects of DLK are independent of proteolytic cleavage of the molecule and require its expression as a transmembrane protein. The intracellular portion of the molecule is critical to inhibition of differentiation but not to proliferation inhibition. In contrast, proteolytic release of the extracellular portion of the molecule may stimulate cell proliferation but does not affect differentiation. Further investigation of the role of DLK overexpression in abnormal hematopoiesis in MDS is warranted.

Cooling, cryoprotectant and hypersaline sensitivity of penaeid shrimp embryos and nauplius larvae

The sensitivity of embryos of the penaeid shrimp, Trachypenaeus byrdi, to cooling, cryoprotectant exposure (dimethyl sulfoxide : DMSO, sucrose, methanol and glycerol), and hypersaline treatment was assessed in order to gain basic knowledge for cryopreservation procedures. In addition, cooling and DMSO exposure was evaluated in Penaeus stylirostris and T. byrdi nauplii. Morulae and advanced embryos (setae development stage) showed tolerance to cooling at 10°C, but were very sensitive to 0°C exposure. Methanol exposure at 12°C up to 2 M, was non-toxic for advanced embryos. DMSO toxicity was intermediate; no statistical decrease in survival ( P>0.05) was measured at 0.5 M. Sucrose and glycerol were toxic to both embryo stages over 0.25 and 0.5 M, respectively. Morulae were more resistant to hypersaline treatment at 55 ppt than advanced embryos. Nauplii showed a better tolerance to cooling and DMSO exposure than embryos. These findings are being applied to develop a cryogenic protocol for penaeid embryos.

The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present

When mouse ovulated oocytes were exposed to 1.5 M-dimethylsulphoxide (DMSO) the resultant hardening of the zona pellucida was not a direct effect but required the presence of an oocyte. The hardening of the zona pellucida when zonae used were aged in vitro was also dependent upon the presence of the oocyte. Protocols of DMSO exposure that induce zona-hardening also caused depletion of the numbers of cortical granules underlying the oocyte surface, whereas protocols without effect on the zona did not reduce significantly the cortical granule count. It is proposed that the effects of DMSO may be mediated by a release of cortical granule contents.