Distinguishing between ante factum and post factum properties of animal cell lines and demonstrating their use in grouping ray-finned fish cell lines into invitromes.

In this review, animal cell lines are considered to have two classes of attributes: "before-the-fact" (ante factum) and "after-the-fact" (post factum) properties. Fish cell lines from Actinopterygii (ray-finned fishes) are used to illustrate this distinction and to demonstrate how these properties can be used in various ways to categorize cell lines into groups or invitromes. Before-the-fact properties are set at initiation and are properties of the sample and species from which the cell line arose and of the scientist(s) who developed the cell line. On the basis of the Actinopterygii sample, invitromes exist for embryos, larvae, juveniles, adults, and spawning fish, and for most solid organs but rarely for biological fluids. For species, invitromes exist for only a small fraction of the Actinopterygii total. As to their development, scientists from around the world have contributed to invitromes. By contrast, after-the-fact properties are limitless and become apparent during development, characterization, use, and storage of the cell line. For ray-finned invitromes, cell lines appear to acquire immortality during development, are characterized poorly for differentiation potential, have numerous uses, and are stored formally only sporadically. As an example of applying these principles to a specific organ, the skeletal muscle invitrome is used. For ante factum properties, the cell lines are mainly from trunk muscle of economically important fish from 11 orders, 15 families, 19 genera, and 21 species of ray-finned fishes. For post factum properties, fibroblast-like and myogenic cell lines have been described but epithelial-like FHM is most widely used and curated. Considering cell lines by their before- and after-the-fact properties should facilitate integration of new cell lines into the literature and help incorporate the discipline of cell biology into other research areas, particularly the natural history of fishes.

A continuous myofibroblast precursor cell line from the tail muscle of Australasian snapper (Chrysophrys auratus) that responds to transforming growth factor beta and fibroblast growth factor.

Chrysophrys auratus (Australasian snapper) is one of the largest and most valuable finfish from capture fisheries in New Zealand, yet no cell lines from this species are reported in the scientific literature. Here, we describe a muscle-derived cell line initiated from the tail of a juvenile snapper which has been designated CAtmus1PFR (Chrysophrys auratus, tail muscle, Plant & Food Research). The cell line has been passaged over 100 times in 3 years and is considered immortal. Cells are reliant on serum supplementation for proliferation and exhibit a broad thermal profile comparable to the eurythermic nature of C. auratus in vivo. The impact of exogenous growth factors, including insulin-like growth factors I and II (IGF-I and IGF-II), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGFß), on cell morphology and proliferation was investigated. Insulin-like growth factors acted as mitogens and had minimal effect on cell morphology. TGFß exposure resulted in CAtmus1PFR exhibiting a myofibroblast morphology becoming enlarged with actin bundling. This differentiation was confirmed through the expression of smooth muscle actin (sma), an increase in type 1 collagen (col1a) expression, and a loss of motility. Expression of col1a and sma was decreased when cells were exposed to bFGF, and no actin bundling was observed. These data indicate that CAtmus1PFR may be myofibroblastic precursor cells descending from mesenchymal progenitor cells present in the tail muscle myosepta.

New body mass index for predicting prognosis in patients with antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVES: Body mass index (BMI) is a known indicator of all-cause mortality. However, conventional BMI does not reflect the three-dimensional human body. To overcome this limitation, a new BMI has been proposed that provides a closer approximation of real human body shape. This study investigated the associations between the new BMI and poor outcomes in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). METHOD: We retrospectively reviewed the medical records of 242 patients with AAV in a single tertiary medical center. Based on the new BMI, the patients were categorized into four groups: underweight (<18.5 kg/m2.5 ), healthy weight (18.5 to <25.0 kg/m2.5 ), overweight (25.0 to <30.0 kg/m2.5 ), and obese (≥30.0 kg/m2.5 ). The association among the new BMI and death, relapse, end-stage renal disease (ESRD) development, cerebrovascular accident, and cardiovascular disease was analyzed. RESULTS: The underweight group, according to the new BMI, had higher hazard ratios (HRs) for all-cause mortality (HR: 3.180, 95% confidence interval [CI]: 1.134-8.922, p = 0.028), relapse (HR: 2.141, 95% CI: 1.019-4.368, p = 0.036), and ESRD development (HR: 2.729, 95% CI: 1.190-6.259, p = 0.018) than the healthy weight group. However, according to the conventional BMI, there were no differences in the risks for all poor outcomes between the underweight and healthy weight groups. Multivariate logistic regression analysis demonstrated that being underweight, according to the new BMI, was an independent risk factor for all-cause mortality (HR: 5.285; 95% CI: 1.468-19.018; p = 0.011). CONCLUSION: Being underweight, according to the new BMI, is associated with poor outcomes in patients with AAV.

Integrity and wound healing of rainbow trout intestinal epithelial cell sheets at hypo-, normo-, and hyper-thermic temperatures.

How temperature influences fish physiological systems, such as the intestinal barrier, is important for understanding and alleviating the impact of global warming on fish and aquaculture. Monolayers of the rainbow trout cell line, RTgutGC, with or without linear 500 µm wide gaps (wounds) were the in vitro models used to study the integrity and healing of intestinal epithelial sheets at different temperatures. Cultures at hypothermic (4 °C) or hyperthermic (≥ 26 °C) temperatures were compared to normothermic control cultures (18-22 °C). Monolayers remained intact for at least a week at temperatures from 4 to 28 °C, but had lost their integrity after 3 h at 32 °C as the cells pulled away from one another and from the plastic surface. F-actin appeared as prominent stress fibers in cells at 28 °C and as blobs in cells at 32 °C. At normothermia and at 26 °C, cells migrated as sheets into the gaps and closed (healed) the gaps within 5-6 days. By contrast, wounds took 14 days to heal at 4 °C. At 28 °C some cells migrated into the gap in the first few days but mainly as single cells rather than collectively and wounds never healed. When monolayers with wounds were challenged at 32 °C for 3 h and returned to 18-22 °C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26 °C for 24 h and challenged at 32 °C for 3 h prior to being placed at 18-22 °C, cell shape and actin cytoskeleton were maintained, and wounds were healed over 6 days. Thus, intestinal epithelial cells become thermostabilized for shape, cytoskeleton and migration by a prior heat exposure.

VER-155008 induced Hsp70 proteins expression in fish cell cultures while impeding replication of two RNA viruses.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 µM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Effect of selenomethionine on cell viability and heat shock protein 70 levels in rainbow trout intestinal epithelial cells at hypo-, normo-, and hyper-thermic temperatures.

As global warming and environmental pollution modify aquatic environments, the thermal biology of fish could be affected by interactions between temperature and pollutants, such as selenium (Se). Therefore, selenomethionine (SeMet) was studied for effects on cell viability and on heat shock protein 70 (HSP70) levels in the rainbow trout intestinal epithelial cell, RTgutGC, at hypothermic (4 °C), normothermic (14 and 18 °C) and hyperthermic (26 °C) temperatures. RTgutGC cultures remained viable for at least a week at all temperatures, although energy metabolism as measured with Alamar Blue (resazurin) was appreciably diminished at 4 °C. Over a 7-day incubation, HSP 70 levels in cultures remained steady at 4 °C, declined at 18 °C, and increased slightly at 26 °C. When 125 µM SeMet was present, cultures remained viable and HSP70 levels were neither increased nor decreased relative to control cultures, regardless of the temperature. With 500 and 1000 µM SeMet, cell viability was profoundly impaired after 7 days in cultures at 14, 18 and 26 °C but was unchanged at 4 °C. Overall the results suggest that only hypothermia modulated the response of rainbow trout cells to SeMet.

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua.

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno-positive results for vimentin, α-smooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua-associated xenoma-like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.

Responses of rainbow trout intestinal epithelial cells to different kinds of nutritional deprivation.

In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz's basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.

Responses of an American eel brain endothelial-like cell line to selenium deprivation and to selenite, selenate, and selenomethionine additions in different exposure media.

The effect of selenium deprivation and addition on the American eel brain endothelial cell line (eelB) was studied in three exposure media: complete growth medium (L15/FBS), serum-free medium (L15), and minimal medium (L15/ex). L15/ex contains only galactose and pyruvate and allowed the deprivation of selenium on cells to be studied. In L15/ex, without any obvious source of selenium, eelB cells survived for at least 7 d, formed capillary-like structures (CLS) on Matrigel, and migrated to heal wounds. Three selenium compounds were added to cultures: selenite, selenate, and selenomethionine (SeMet). Adding selenite or selenate to eelB cell cultures for 24 h caused dose-dependent declines in cell viability, regardless of the exposure media. Although varying with exposure media and viability end point, selenite was approximately 70-fold more cytotoxic than selenate. By contrast, 24 h exposures to either DL- or L-SeMet in the three media caused little or no cytotoxicity. However for 7 d exposures in L15/ex, DL- and L-SeMet were very cytotoxic, even at the lowest tested concentration of 31 µM. By contrast in L15 and L15/FBS, cytotoxicity was only observed with 500 and 1000 µM L-SeMet. In L15/FBS, eelB continued to migrate and form CLS in the presence of SeMet but at 500 µM, cell migration appeared stimulated. As judged from a colony-forming assay over 14 d in L15/FBS, 500 and 1000 µM DL- and L-SeMet inhibited cell proliferation. Overall, the responses of eel cells to selenium depended on the selenium form, concentration, and exposure media, with responses to SeMet being most dependent on exposure media.

Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout.

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18-22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.

HandKAchip - Hands-free killing assay on a chip.

Small animals such as the roundworm C. elegans are excellent models for studying bacterial infection and host response, as well as for genetic and chemical screens. A key methodology is the killing assay, in which the number of surviving animals is tracked as a function of the time post infection. This is a labor-intensive procedure, prone to human error and subjective choices, and often involves undesired perturbation to the animals and their environment. In addition, the survival of animals is just one aspect of a multi-dimensional complex biological process. Here we report a microfluidic-based approach for performing killing assays in worms, compatible with standard assays performed on solid media. In addition to providing accurate and reproducible survival curves at a considerably reduced labor, this approach allows acquisition of a multitude of quantitative data with minimal undesired perturbations. These measurements are obtained automatically at a worm-by-worm resolution using a custom image processing workflow. The proposed approach is simple, scalable, and extendable, and is significantly more economical than standard manual protocols.

Development of a cell line from the American eel brain expressing endothelial cell properties.

A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated ß-galactosidase (SA ß-Gal) activity.

Development and characterization of an endothelial cell line from the bulbus arteriosus of walleye, Sander vitreus.

A cell line has been developed from the bulbus arteriosus (BA) of the walleye (WE), Sander vitreus (Mitchill), and is termed WEBA. WEBA produced collagen I, and when held at confluency for days or weeks, spontaneously formed capillary-like tubes. WEBA cells bound fluorescently-labeled Ulex europaeus lectin agglutinin I (UEA-1), took up acetylated low density lipoprotein (Ac-LDL), were stained for von Willebrand factor (vWF), and produced nitric oxide (NO). The cytoskeleton consisted at least of α- and ß-tubulin, vimentin, and actin, with the actin organized into circumferential bundles. Immunofluorescence staining revealed at least two tight junction proteins, zonula occludens-1 (ZO-1) and claudin 3. Together these results suggest that WEBA is an endothelial cell line. Relatively high doses of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) induced cytochrome P4501A (CYP1A) protein and 7-ethoxyresorufin o-deethylase (EROD) activity in WEBA. As one of the first fish endothelial and BA cell lines, WEBA should be useful in many disciplines in which the teleost cardiovascular system is a focus.

Senescence-associated ß-galactosidase staining in fish cell lines and primary cultures from several tissues and species, including rainbow trout coelomic fluid and milt.

Cell lines and primary cultures from several teleost tissues and species were stained for senescence-associated ß-galactosidase (SA ß-Gal), revealing four general outcomes. (1) For long-standing fish cell lines that can be considered immortal, little or no SA ß-Gal staining was observed, regardless of the culture conditions. (2) For a new walleye cell line from the bulbus arteriosus (WEBA), most cells stained for SA ß-Gal even after 40 passages. This suggested that high SA ß-Gal activity was a unique property of WEBA, perhaps reflecting their endothelial character, rather than cellular senescence. (3) For cell lines developed from the walleye caudal fin and from somatic cells in rainbow trout coelomic fluid, no SA ß-Gal staining was observed in the earliest cultures to over 70 passages later. This suggested that cells from these anatomical sites do not undergo senescence in vitro. (4) By contrast, for cell lines developed from the walleye brain and from somatic cells in rainbow trout milt, most cells in the early-stage cultures stained for SA ß-Gal, but as these were developed into cell lines, SA ß-Gal-negative cells became dominant. This suggested that if cellular senescence occurred in vitro, this happened early in these cultures and subsequently a few SA ß-Gal-negative cells went onto to form the cell line. Overall, the presence of SA ß-Gal-positive cells in cultures could be interpreted in several ways, whereas their absence predicted that in these cultures, cells would proliferate indefinitely.

Proteomic responses in the gills of fathead minnows (Pimephales promelas, Rafinesque, 1820) after 6 months and 2 years of continuous exposure to environmentally relevant dietary 226Ra.

PURPOSE: To investigate changes in the gill proteome of fathead minnows continually fed an environmentally relevant dietary dose of (226)Ra for 2 years. METHODS: The fish were fed a commercial diet containing 10 mBq-10 Bq (226)Ra g(-1). After 6 months and 2 years the gill proteome was analyzed by two-dimensional electrophoresis (2-DE). Protein spots which exhibited a significant change were identified using mass spectrometry. RESULTS: Six proteins were found to be increased: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, protein disulphide isomerase precursor, ATP synthase, glial cell fibrillary protein (GFAP), apolipoprotein A1 (ApoA1). One protein was found to be decreased; malate dehydrogenase. The majority of these changes occurred predominantly at the lowest (226)Ra doses, within 6 months and were maintained for 2 years. CONCLUSIONS: These proteomic changes suggested an adaptive or protective response to radiation induced reactive oxygen species (ROS). Increased GFAP indicated the induction of oxidative stress. Increased GAPDH and enolase indicated enhanced ROS scavenging from glycolytic metabolites. Increased protein disulphide isomerase precursor indicated an enhanced source of radioprotective thiols. Decreased malate dehydrogenase indicated enhanced ROS scavenging within the mitochondria. Increased ATP synthase indicated enhanced protection of healthy cells and increased ApoA1 indicated enhanced protection of the gill lamellae.

Derivation of a continuous myogenic cell culture from an embryo of common killifish, Fundulus heteroclitus.

The common killifish or mummichog (Fundulus heteroclitus) is an estuarine teleost increasingly used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions and ease of maintenance has made them popular aquatic research organisms. Scientific advances with most popular model organisms have been assisted with the availability of continuous cell lines; however, cell lines from F. heteroclitus appear to be unavailable. The development of a killifish cell line, KFE-5, derived from the mid trunk region of a late stage embryo is described here. KFE-5 grows well in Leibovitz's L-15 media with 10% fetal bovine serum (FBS). This cell line has been passaged over 60 times in a span of three years, and cells at various passages have been successfully cryopreserved and thawed. The cells are mostly fibroblastic but contain myogenic cells that differentiate into mono-, bi- and multi-nucleated striated myocytes. Immunofluorescence detection of muscle specific antigens such as α-actinin, desmin, and myosin confirms KFE-5 as a myogenic cell line. KFE-5 has a temperature preference for 26-28°C and has been shown to withstand temperatures up to 37°C. The cell line responds to chemical signals including growth factors, hormones and extracellular matrix components. KFE-5 could thus be useful not only for mummichog's thermobiology but also for studies in fish muscle physiology and development.

Cytotoxicity evaluation of silica nanoparticles using fish cell lines.

Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 µg/mL were needed to achieve 24 h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays.

The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD's fish toxicity testing framework No. 171.

Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co-operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost-effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to chemical contaminants. This fluorometric assay involves: (1) alamar blue for metabolic activity, (2) CFDA-AM for membrane integrity, and (3) neutral red for lysosomal function. These protocols are conveniently performed in semi-unison within the same multiwell plates and read at three different wavelengths. Detailed step-by-step descriptions of the assays, parameters to consider, troubleshooting, and guidelines for data interpretation are provided as essential tools for investigating environmental aquatic contaminants at the cellular level.

Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines.

Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R (2) between 0.4519 and 0.6171; p<0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.

Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells.

Rainbow trout gill epithelial cells (RTgill-W1) are used in a cell-based biosensor that can respond within one hour to toxic chemicals that have the potential to contaminate drinking water supplies. RTgill-W1 cells seeded on enclosed fluidic biochips and monitored using electric cell-substrate impedance sensing (ECIS) technology responded to 18 out of the 18 toxic chemicals tested within one hour of exposure. Nine of these chemical responses were within established concentration ranges specified by the U.S. Army for comparison of toxicity sensors for field application. The RTgill-W1 cells remain viable on the biochips at ambient carbon dioxide levels at 6°C for 78weeks without media changes. RTgill-W1 biochips stored in this manner were challenged with 9.4µM sodium pentachlorophenate (PCP), a benchmark toxicant, and impedance responses were significant (p<0.001) for all storage times tested. This poikilothermic cell line has toxicant sensitivity comparable to a mammalian cell line (bovine lung microvessel endothelial cells (BLMVECs)) that was tested on fluidic biochips with the same chemicals. In order to remain viable, the BLMVEC biochips required media replenishments 3 times per week while being maintained at 37°C. The ability of RTgill-W1 biochips to maintain monolayer integrity without media replenishments for 78weeks, combined with their chemical sensitivity and rapid response time, make them excellent candidates for use in low cost, maintenance-free field-portable biosensors.