- Email: [email protected]
Cell culture-based biosensing techniques for detecting toxicity in water
Available online at www.sciencedirect.com
ScienceDirect Cell culture-based biosensing techniques for detecting toxicity in water Lu Tan1 and Kristin Schirmer1,2,3 The significant increase of contaminants entering fresh water bodies calls for the development of rapid and reliable methods to monitor the aquatic environment and to detect water toxicity. Cell culture-based biosensing techniques utilise the overall cytotoxic response to external stimuli, mediated by a transduced signal, to specify the toxicity of aqueous samples. These biosensing techniques can effectively indicate water toxicity for human safety and aquatic organism health. In this review we account for the recent developments of the mainstream cell culture-based biosensing techniques for water quality evaluation, discuss their key features, potentials and limitations, and outline the future prospects of their development. Addresses 1 Department of Environmental Toxicology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, U¨berlandstrasse 133, 8600 Du¨bendorf, Switzerland 2 School of Architecture, Civil and Environmental Engineering (ENAC), EPF Lausanne, 1015 Lausanne, Switzerland 3 Institute of Biogeochemistry and Pollutant Dynamics, ETH Zu¨rich, 8092 Zu¨rich, Switzerland Corresponding author: Schirmer, Kristin ([email protected])
Current Opinion in Biotechnology 2017, 45:59–68 This review comes from a themed issue on Environmental biotechnology Edited by Jan Roelof Van Der Meer and Man Bock Gu For a complete overview see the Issue and the Editorial Available online 27th January 2017 http://dx.doi.org/10.1016/j.copbio.2016.11.026 0958-1669/ã 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).
Introduction Today in both developing and industrialised countries, an increasing number of contaminants, pertaining to 300 million tons of industrial chemicals and 145 million tons of fertilisers and pesticides produced worldwide annually, are entering the water bodies [1]. They are impairing ecosystems, and in turn, threatening the water security of the world’s population [2,3]. In the US, about half of the assessed stream miles and lake acres, and one third of the assessed bay and estuarine square miles are classified as polluted; in China, the majority of rivers and lakes, and all of its coastal waters are polluted [4]. In the European Union, only a fraction of the www.sciencedirect.com
>100 000 EINECS (European INventory of Existing Commercial chemical Substances) registered chemicals have their assessment reports available in the OECD (Organisation for Economic Co-operation and Development) Existing Chemicals Database [5]. The effects of most chemicals on the ecosystem are largely unknown. Increasing initiatives and legislative actions for water pollution control have been adopted [6]; the Water Framework Directive (WFD) has been established to govern the European water policy, which places aquatic ecology at the basis of water management decisions [7,8]. The demand for restoring the water status calls for rapid, reliable and economic methods for water monitoring and chemical toxicological impact identification and assessment [9]. The fish acute toxicity test (OECD test guideline 203) is the most widely applied assay for aquatic environmental risk assessment, where 42–60 fish are exposed for 96 hours and death is used as end point. The latest available data showed that, in the EU alone, 179 083 fish were used in regulatory toxicological and safety evaluations in 2011 [10], whilst an estimated three million fish are used in North America for effluent testing each year [11]. Besides being technically challenging, time-consuming and costly, fish tests also raise ethical concerns of animal welfare; thus there is a strong demand for replacement, reduction and refinement approaches [12,13,14,15]. The past years have seen many efforts to develop alternatives for animal testing, and the most extensively explored are the in vitro cell models [11,16,17,18,19]. A variety of fish cell-based methods have been developed in testing of chemical and whole water effluents, and shown to be effective, reproducible and economic [12]. Recently, close sensitivity and correlation between rainbow trout gill cell (RTgill-W1) cytotoxicity and acute fish toxicity have been demonstrated [11], indicating the predictive power of cell models and their promising application as alternatives for animal experimentation. It is generally accepted that the replacement of whole animal tests by a single alternative method is difficult, but the use of alternatives to significantly reduce animal testing is desired and possible [14]. Biosensors, incorporating cell-mediated biological recognition systems and signal transducing elements, offer even more advantages of rapid response, portability, field applicability, being real-time and cost-effective, compared to the conventional cell-based bioassays [19,20], and thus are excellent choices for water status monitoring Current Opinion in Biotechnology 2017, 45:59–68
60 Environmental biotechnology
and chemical toxicity detection. Most ‘cell-based’ biosensors refer to microbes, which are not within the scope of this review. The term ‘cell’ in the following context all refers to vertebrate cells originating from different tissues. Biosensor developments for water toxicity detection often originate from either human safety (drinking water quality) or ecotoxicological concerns (water quality for biota and the aquatic environment). However, as many cell-based biosensors can be used for water quality evaluation of both human and ecosystem health, boundaries for their applications are rather blurry. Therefore, in the techniques reviewed here, we do not distinguish whether a method was originally developed for human safety or
ecotoxicology. Figure 1 summarises the basics of cell culture-based biosensors. In the review we will highlight the recent major developments of cell-based biosensing methods for water toxicity, and discuss their features, potentials, limitations and further developments.
Cell-based biosensing techniques Electric cell-substrate impedance sensing (ECIS)
The most vibrant research activity in cell-based biosensing for water assessment builds on the Electric Cellsubstrate Impedance Sensing [21] (ECIS) technology. ECIS utilises cell culture medium as electrolyte with cells grown on an electrode-embedded culturing surface.
Figure 1
Detectable contaminants
Contaminants in water
Cells
Industrial chemicals
Heavy metals
Pharmaceuticals & pesticides
Pathogens & their toxins
PAHs
Chem. mixtures in fresh- & wastewater
Toxicity manifestation
Etc.
Applications Etc. Detachment Morphology change
Death
Pigment translocation
On-site chemical impact assessment
Ecosystem conservation
Signal transduction and toxicity indication Water monitoring
Contaminated
or
Not Contaminated
Early warning system
Current Opinion in Biotechnology
Cell-based biosensors for detecting toxicity in water. Known or unknown contaminants in water elicit various toxic effects in cells, which can be manifested as cell detachment, morphology change, cell death, and in case of chromatophores, the translocation of pigments. These cytotoxic signals can be transduced in biosensors, by, for example cell impedance or pigment tracking, to an integrated receiving end to indicate the toxicity. Cell-based biosensors are able to detect a broad spectrum of substances and can be applied in many different areas of toxicity detection in water. Some of the applications may share some common aims (overlapping circles).
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 61
By applying a very low, non-invasive alternating current, the cell electric impedance can be measured to indicate the cell status including growth, migration, morphology, cell-to-matrix and cell-to-cell interactions [22]. Research using ECIS technology to evaluate water quality has been consistently led by the US Army Center for Environmental Health Research for the purpose of developing a fielduse device for Army personnel deployed around the world to rapidly test drinking water for possible chemical contaminations [23,24]. The decade-long development route [23,24,25,26,27,28] (Table 1, ECIS) started with an initial testing of a dozen of toxic industrial chemicals using 10 different sensors, with results putting ECIS among the top performing technology [23]. The openwell ECIS platform was combined with an enzyme-based acetylcholinesterase inhibition sensor, to form the Environmental Sentinel Biomonitor (ESB) system [24]. The ECIS platform was further modified and refined to a portable automated device incorporating a self-contained disposable media delivery system [25]. By this point, cells used were mammalian cells that required tightly controlled maintenance condition. This new system enabled the long-term keeping of the mammalian cells (up to 16 weeks of responsiveness) through the automated media delivery system, which was a significant step forward. Further milestone development adopted the rainbow trout gill cells (RTgill-W1) that showed comparable performance as mammalian cells, and significantly more robustness and simple maintenance with a tested 39 weeks of responsiveness [26]. This study fulfilled the prospect of using the system as a portable, low-cost, lowmaintenance biosensor. Follow-up researches [27,28] refined the procedural steps and added a hand-held reader displaying ‘contaminated’ or ‘not contaminated’ result for non-expert users, readying the device for military and municipal field-use. Figure 2a,b show the RTgill-W1 cells and the ECIS biochip. Although the systematic development procedure solved many technical issues, limitations inherent to the cell line, such as the insensitivity to certain classes of stimuli could not be overcome using the ECIS biosensor alone. Other impedance-based methods
Several commercial systems based on cell impedance, such as xCELLigence (ACEA Biosciences, USA) and Bionas (Bionas, Germany), often applied in pharmaceutical research, have also been used in assessing toxicity in water. xCELLigence (Table 1, xCELLigence) measures cell impedance represented as Cell Index (CI) that reflects cell growth, death, adhesion and morphology [29]. The larger the CI value, the more viable cells are attached to the system. Perturbation by external stimuli can be detected via the change of CI values. Eight mammalian cell lines and a rainbow trout skin derived cell line were tested using xCELLigence in various studies [29,30,31]. Although there were variations comparing the impedance method with viability www.sciencedirect.com
assays such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), and also differences between cell lines within the same assay, the xCELLigence system was generally effective in reacting to purposely spiked toxicants in water [31] as well as to real water samples [29]. Bionas (Table 1, Bionas) is a multiparametric system integrating three parameter read-outs: cell impedance, acidification (metabolism) and oxygen consumption (respiration); five mammalian cell lines were tested with Bionas [30,32]. The integrative use of multiple parameters, particularly oxygen consumption, enabled more rapid detection of toxicants [30]. The system was suitable for detecting a broad range of substances including heavy metals, pharmaceuticals, neurotoxins and contaminants in wastewater [32]. Another multiparametric biosensor (Table 1, ECIS/QCM) with impedance and mass-sensing through a Quartz Crystal Microbalance (QCM) resonator was developed and shown to be effective in fast detection of various chemicals with increased accuracy [33,34,35]. Chromatophore-based approaches
Chromatophores are neurone-like pigmented cells found in amphibians, fish and reptiles, and are able to respond to stimuli with changing pigment distribution. The pigment aggregation and dispersion are mainly mediated by Gprotein linked signal transduction involving phosphorylation events and motor protein mobilisation to translocate the pigment granules [36]. In chromatophore-based water toxicity biosensing (Table 2, Chromatophore-based approaches), the most commonly applied chromatophores originated from Siamese fighting fish [36,37,38,39,40], and also from Chinook salmon [41,42], Nile tilapia [23] and African clawed frog [43]. These cells are shown to respond to live pathogenic microbes or microbial toxins and a wide range of chemicals including heavy metals, organophosphate pesticides and polynuclear aromatic hydrocarbons (PAHs) [23,36,40]. Although the fish species inhabit different ecological niches, their responses to external stimuli are highly conserved [41,42], suggesting that similar cellular mechanisms govern the change of pigment distribution. An example of chromatophore pigment relocation is shown in Figure 2c. This technique was pioneered by integrating light source, camera and lens, monitor and keyboard and an image processing software into a portable device [36]. Further chromatophore-based biosensing studies adopted similar setup components [23,37]. Quantification methods have been a key issue in interpreting chromatophore test results, mainly caused by the heterogeneity of fast- and slowresponding cells, the variation in number and density of chromatophore samples, and the ambiguity of subtle changes [36,38,40]. Modelling and refinement in quantitative analysis are often necessary; commonly applied methods quantify changes of cell area covered by pigment [23,37,38] using photographic recording of cells upon a fixed field of view containing the same number Current Opinion in Biotechnology 2017, 45:59–68
62 Environmental biotechnology
Table 1 Impedance-based biosensing techniques for water toxicity detection ECIS Cell
Cell type
Origin
Biosensing mechanism
Tested substances
Test time a
Longevity
Realtime
Ref.
BPAEC
Mammalian
x
[23]
1 hour
30 days
x
[24]
BPAEC
Mammalian
1 hour
9 days
x
[25]
BLMVEC
Mammalian
Bovine lung microvessel endothelial cells
1 hour
16 weeks
x
[25]
RTgill-W1
Fish
1 hour
39 weeks
x
[26]
BLMVEC
Mammalian
16 weeks
x
[26]
Fish
18 chemicals including aldicarb, ammonia, paraquat, and so on 18 chemicals including aldicarb, ammonia, paraquat, and so on
1 hour
RTgill-W1
1 hour
N. M.
x
[27]
RTgill-W1
Fish
Rainbow trout (Oncorhynchus mykiss) gill cells Bovine lung microvessel endothelial cells Rainbow trout (Oncorhynchus mykiss) gill cells Rainbow trout (Oncorhynchus mykiss) gill cells
12 chemicals incl. aldicarb, ammonia, paraquat, and so on 12 chemicals incl. aldicarb, ammonia, paraquat, and so on Five chemicals incl. phenol, potassium cyanide, pentachlorophenate, and so on Five chemicals incl. phenol, potassium cyanide, pentachlorophenate, and so on 18 chemicals including aldicarb, ammonia, paraquat, and so on
N. M. b
Mammalian
Changes in cell-substrate impedance after perturbation by chemicals
1 hour
BLMVEC
Bovine pulmonary artery endothelial cells Bovine lung microvessel endothelial cells Bovine pulmonary artery endothelial cells
13 chemicals including aldicarb, ammonia, paraquat, and so on
1 hour
N. M.
x
[28]
Longevity
Realtime
Ref.
xCELLigence Cell
Cell type
Origin
Biosensing mechanism
V79
Mammalian
NIH-3T3
Mammalian
Impedance measured as cell index
RAsd CEsd
Mammalian Mammalian
OMYsd
Fish
A549
Mammalian
ACHN
Mammalian
HepG2
Mammalian
SK-N-SH
Mammalian
Chinese hamster lung cells Mouse embryo fibroblast cells Rat skin derived cells Human skin derived cells Rainbow trout (Oncorhynchus mykiss) skin derived cells Human lung epithelial cells Human kidney derived cells Human hepatoblastoma cells Human neuroblastoma cells
Tested substances
Test time
N. M.
x
[30]
Copper sulfate pentahydrate
Several to 24 hours 24 hours
N. M.
x
[31]
Copper sulfate pentahydrate Copper sulfate pentahydrate
24 hours 24 hours
N. M. N. M.
x x
[31] [31]
Copper sulfate pentahydrate
24 hours
N. M.
x
[31]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Chromium
Bionas Cell
Cell type
Origin
V79
Mammalian
HT-29
Mammalian
L6
Mammalian
Chinese hamster lung cells Human colon adenocarcinoma Rat skeletal muscle
HepG2
Mammalian
V79
Mammalian
Biosensing mechanism Multiparametric: changes in impedance, pH and oxygen consumption
Human hepatoblastoma cells Chinese hamster lung cells
Current Opinion in Biotechnology 2017, 45:59–68
Tested substances Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Chromium
Test time
Longevity
Realtime
Ref.
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
Several to 24 hours
N. M.
x
[30]
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 63
Table 1 (Continued ) ECIS/QCM Cell
Cell type
BAEC
Mammalian
BAEC
Mammalian
BAEC
Mammalian
Origin Bovine aortic endothelial cells Bovine aortic endothelial cells Bovine aortic endothelial cells
Biosensing mechanism Multiparametric: changes in impedance and resonance frequency through mass sensor
Tested substances
Test time
Longevity
Realtime
Ref.
Ammonia
1 hour
34 days
x
[33]
Cell behaviour characterisation Ammonia, nicotine and aldicarb
Several days
6 weeks
x
[34]
Up to 1 hour
30 days
x
[35]
References in each category are listed by time of publication (most recent at the end). Is taken from method description if available, or from information in diagrams if not specified in method. Response time is commonly within or close to test time. b N. M. = not mentioned. This information may be available in other studies that tested the longevity for the same cell line under similar conditions. a
of cells. That chromatophores are primary cells and unable to continuously propagate in culture, adds to the complexity of data interpretation and reproducibility, and limits their application for routine water toxicity evaluation.
Besides the impedance- and chromatophore-based approaches, several other techniques were also tested in water toxicity detection. These researches utilised distinct biosensing mechanisms [23,44,45,46] (details in Table 2, Other approaches) but were more fragmented,
Figure 2
(a)
(b)
250 µm
50 µm (c)
Current Opinion in Biotechnology
Examples of cells in cell-based biosensors for water toxicity detection. (a) Live cell staining of the RTgill-W1 cells, where nuclei (blue), cell membrane (green), and lysosome (magenta) are visible. (b) With permission from Ref. [26], ECIS biochip with RTgill-W1 cells. (c) With permission from Ref. [40], aggregation of pigment (right) in chromatophores (untreated, left) of Siamese fighting fish after treatment with norepinephrine (1 mg/ml). www.sciencedirect.com
Current Opinion in Biotechnology 2017, 45:59–68
64 Environmental biotechnology
Table 2 Chromatophore-based biosensing techniques and other approaches for water toxicity detection Chromatophore-based approaches Cell
Cell type
Origin
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Nile tilapia (Oreochromis niloticus)
Melanophores
Amphibian
Melanophores
Fish
Melanophores
Fish
Melanophores from African clawed frog (Xenopus laevis) Chinook salmon (Oncorhynchus tshawytscha) Chinook salmon (Oncorhynchus tshawytscha)
Biosensing mechanism Translocation of cellular pigments upon perturbation of cellular processes Translocation of cellular pigments upon perturbation of cellular processes Changes in the total area of colour in the cells Changes in pigment covered area in cells immobilised to beads Changes in light transmitted through the cell area of the chromatophores Changes in chromatophore distribution Changes in light absorption by the cells after chemical exposure Changes in cell pigments Changes in cell pigments
Test time a
Longevity
Ref.
Bacterial toxins and chemicals such as PAHs
1 hour
3 months
[36]
Various cellular effectors and bacterial toxins
1 hour
N. M.
Bacterial toxins
20 min
13 weeks
[39]
Clonidine
6 min
2–4 weeks
[38]
Clonidine and cirazoline
5 min
Up to 1 month
[40]
12 chemicals incl. aldicarb, ammonia, paraquat, and so on 12 chemicals incl. aldicarb, ammonia, paraquat, and so on Pathogens
1 hour
N. M.
[23]
1 hour
N. M.
[43]
20 min
N. M.
[41]
Environmental toxicants incl. mercuric chloride and sodium arsenite
1 hour
3 weeks
[42]
Tested substances
b
[37]
Other approaches Cell
Cell type
Origin
Biosensing mechanism
Tested substances
Test time
Longevity
Ref.
Epithelioma papulosum cyprini derived from a skin tumour of carp (Cyprinus carpio) Human hepatoblastoma cells
Redox mediated currents generated by metabolising cells when perturbed by chemicals Changes of fluorophore-labelled LDL-update in cells after contaminant exposure Shift in refractive index at the microring resonator surface caused by chemical toxicity Changes in bioluminescence
p-Benzoquinone; 2,6dimethylbenzoquinone
1 hour
N. M.
[44]
12 chemicals incl. aldicarb, ammonia, paraquat, and so on
2 hours
N. M.
[23]
Sodium pentachlorophenate and aldicarb
1 hour
2 days
[45]
Aqueous samples from daily life
30 min
2–3 days
[46]
EPC
Fish
Hep G2
Mammalian
MS1
Mammalian
Mile Sven 1 of pancreas from mouse
Hek293T
Mammalian
Human embryonic kidney cells, engineered, bioluminescent
References in each category are listed by time of publication (most recent at the end). Is taken from method description if available, or from information in diagrams if not specified in method. Response time is commonly within or close to test time. b N. M. = not mentioned. This information may be available in other studies that tested the longevity for the same cell line under similar conditions. a
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 65
involving few precedence or follow-up of development or application.
Key features of cell-based biosensing techniques Choices of cells, longevity and stability
The majority of cell lines used in the mainstream impedance-based water toxicity biosensors are mammalian cells, with the consideration that mammalian cells would better address the toxic responses relevant to human health and safety. However, both mammalian and fish cells were able to respond to contaminants within toxic ranges, and were comparable in relative terms in all available biosensing studies [26,31]. This is largely caused by the fact that cell-based biosensors detect the overall cytotoxic effects, which is essentially a response of basal toxicity common to all cells. However, as the fledgling technology developed to fit practicality for realistic applications, the need for rigorous maintenance and the limited longevity and stability of mammalian cells became a major hurdle. The earlier ECIS-based systems used the relatively robust mammalian cells from the bovine pulmonary tissues [23,24,25,26,33,34,35] which were shown to stay viable and responsive for one [33] to three months [25]. Nevertheless, these cells require medium replenishment several times a week and maintenance at 37 C and 5% of CO2, which significantly reduces their applicability in a portable field-device. The later development of ECIS adopted the rainbow trout gill cells (RTgill-W1) [47], which is a continuous cell line and can survive across a wide range of temperature (4–24 C) with optimal growth condition at 20 C and no requirement on CO2 [48]. This cell line is one of the most extensively researched and applied models in the context of alternatives to fish tests and water toxicity [11,48,49,50,51,52]. Its cytotoxicity is closely correlated to fish data; it can be used as a predictor for in vivo acute toxicity [11]. RTgill-W1 can be exposed in serum-free conditions to eliminate interference of serum proteins with toxicants [11]. These cells do not require media change at low-temperature storage condition, and have been tested with toxicant responses after about nine months of storage [26]. These characteristics clearly advantage fish cells over mammalian cells for biosensing applications in water. In the chromatophore-based approaches, cells are mainly isolated from fish and have been shown to have an active life of several weeks [38,41] up to three months [36,39]. Unlike the continuous cell lines applied in impedance-based approaches, chromatophores are primary cells and have the inherent limitation in batch-to-batch variability and reproducibility. Detectable substances
Cells used in impedance-based biosensing detect a wide range of toxic substances including heavy metals, industrial chemicals, pharmaceuticals, and chemical mixtures www.sciencedirect.com
in freshwater and wastewater [23,24,25,26,27,28,29, 30,31,32,34,35]. Furthermore, fish cells have been used in water evaluation in conventional bioassays with larger collection of chemicals and environmental samples, including PAHs [53], industrial effluents [16], ammonia [54], diverse organic chemicals [11], and nanoparticles [52]. Such substances and samples should also be able to elicit positive responses on the cell-based impedance biosensing. However, inherent to lacking of receptors in certain tissues, not all cells are sensitive to all types of chemical classes. For example, the neurotoxic chemicals permethrin and lindane were significantly less acutely toxic to RTgill-W1 cells than to fish [11]. Chromatophores have been shown to be sensitive to toxic industrial chemicals and environmental toxicants [23,42,43], live microbial pathogens and bacterial toxins [36,37,39,41]. Response time and real-time testing
Rapid response time is an important criterion for aquatic toxicity biosensing. In the series of ECIS biosensing development, cells responded to many of the tested chemicals within one hour of exposure [23,24,25,26, 27,28]. Monitoring of chemical exposure on the commercial devices xCELLigence and Bionas were usually set to be several to 24 hours [30,31,32], and the freshwater samples to over 100 hours [29]. The chromatophorebased approaches could also detect chemical toxicity in acute exposure of about one hour [42,43]. It needs to be noted that response time is largely determined by the test doses. The criteria in the ECIS studies were set to be between the MEG (Military Exposure Guidelines) and HLC (Human Lethal Concentration) [23,26,27], where the HLC could be one to five orders-of-magnitude higher than the MEG [26]. Within one hour of exposure, toxicant dose was usually much higher than the MEG, and closer to HLC [26]. In the conventional bioassays, however, cells were usually exposed for 24 hours with a range of doses [11]. The higher the toxicant dose, the more rapid the cellular responses are likely to be. Freshwater samples could be much less toxic than spiked and wastewater samples and thus require longer exposure time; for example, a study using freshwater samples carried out the monitoring for several days [29]. Therefore, the term ‘rapid response’ needs always to be interpreted within a context in relation to chemical dosage. In field applications, a ‘rapid response’ would be likely in a chemical spill accident or wastewater, and less likely in monitoring of eventless freshwater bodies in little polluted regions. Real-time monitoring is an advantage of impedancebased biosensing technology. The time-dependent changes of impedance can be recorded throughout chemical exposure. This enables continuous observation of the system, and termination of the tests at any time (i.e. when a positive response is visible). This offers particular advantages in testing samples with unknown substances. Current Opinion in Biotechnology 2017, 45:59–68
66 Environmental biotechnology
Combined and multiparametric biosensors
The testing on overall cytotoxicity can be extended to other, i.e. more specific assays. Two trends dominate the development of complementing systems. One is the combined use of other biosensors, where a battery of multiple sensors provides an even wider range of chemical detection [23]. The other is the integration of several biosensing techniques into one multiparametric device [24,30,32,33,34,35]. The enzyme-based acetylcholinesterase inhibition test was added to ECIS in the ESB system [24,27,28]. The Bionas utilised three parameters including impedance, pH and respiration, and was shown to enable much faster detection of chromium with the respiration assay [30,32]. Moreover, the weight-sensing QCM technique was integrated with ECIS for enhanced sensitivity and accuracy in chemical detection [33,34,35].
Conclusions Important developments took place in recent years in cell-based biosensing techniques for water quality assessment. In future developments, continued exploration in impedance-based approaches using fish cell models, such as RTgill-W1 cells, will boost the field-applicability of cell-based biosensors and bridge the gap in the variety of tested chemicals/samples between conventional bioassays and biosensors. This will improve our understanding of the biosensing mechanism and efficacy, and the approach will also add to the plethora of means in reducing animal usage and finding alternatives to animal testing. Chromatophores offer the distinct direction of rapid biosensing based on optic transduction, but its further development largely depends on overcoming the limitations associated with primary cell cultures, heterogeneity and reproducibility. Since many non-chromatophore, continuous fish cell lines were spontaneously immortalised, the possibilities to immortalise chromatophores and stabilise their variability merit further investigation. Since a single type of cells is not able to respond to all kinds of contaminants, based on the application purpose, combinatorial or multiparametric biosensing can complement the cell-based biosensors and will be a trending focus in biosensor development. The Envirobot [55,56,57], a compartmented autonomous robotic system incorporating various biological sensors based on vertebrate cells, daphnia, amphibian oocytes, microorganism, etc., is a leading example of large-scale multiparametric biosensing for water quality. Although a number of cell culture-based biosensors have been integrated, field testing and actual applications are still limited. The ability to transform a scientific research project to application is the key. This transformation is demanded by the urgent need for effective water monitoring methods, and has seen some endeavours in obtaining product development protection through patent applications [58,59]. In the future, actively bridging the legislative requirements and water initiatives during sensor development, as well as intensified Current Opinion in Biotechnology 2017, 45:59–68
exploration of proprietary rights and commercialisation routes, will significantly help implementing the real-life usage of water toxicity biosensors in various applications.
Acknowledgement The authors acknowledge the financial support from Swiss National Science Foundation Nano-Tera, project Envirobot, grant 20NA21-143082.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Schwarzenbach RP: The challenge of micropollutants in aquatic systems. Science 2006, 313:1072-1077.
2.
Shannon MA, Bohn PW, Elimelech M, Georgiadis JG, Marin˜as BJ, Mayes AM: Science and technology for water purification in the coming decades. Nature 2008, 452:301-310.
3.
Vo¨ro¨smarty CJ, McIntyre PB, Gessner MO, Dudgeon D, Prusevich A, Green P, Glidden S, Bunn SE, Sullivan CA, Liermann CR et al.: Global threats to human water security and river biodiversity. Nature 2010, 467:555-561.
4.
Eltzov E, Marks RS: Whole-cell aquatic biosensors. Anal Bioanal Chem 2011, 400:895-913.
5.
OECD: Status of Chemicals in the Assessment Process. OECD; 2016 http://webnet.oecd.org/hpv/ui/OverallStatus.aspx.
6.
Farre´ M, Barcelo´ D: Biosensors for aquatic toxicology evaluation. In Biosensors for Environmental Monitoring of Aquatic Systems. Edited by Barcelo´ D, Hansen P-D. Berlin, Heidelberg: Springer; 2009:115-160.
7.
Namour P, Lepot M, Jaffrezic-Renault N: Recent trends in monitoring of European Water Framework Directive priority substances using micro-sensors: a 2007–2009 review. Sensors 2010, 10:7947-7978.
8.
Hering D, Borja A, Carstensen J, Carvalho L, Elliott M, Feld CK, Heiskanen A-S, Johnson RK, Moe J, Pont D: The European Water Framework Directive at the age of 10: a critical review of the achievements with recommendations for the future. Sci Total Environ 2010, 408:4007-4019.
9.
Lagarde F, Jaffrezic-Renault N: Cell-based electrochemical biosensors for water quality assessment. Anal Bioanal Chem 2011, 400:947-964.
10. EU: Seventh Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union. European Commission; 2013. 11. Tanneberger K, Kno¨bel M, Busser FJM, Sinnige TL, Hermens JLM, Schirmer K: Predicting fish acute toxicity using a fish gill cell line-based toxicity assay. Environ Sci Technol 2013, 47:1110-1119. The study demonstrated the close correlations betweenin vitro data from fish cells and in vivo acute toxicity of fish, indicating the predictive power of the cell model RTgill-W1 to fish acute toxicity. This provides the basis of using cells in chemical risk evaluation of the aquatic environment. 12. Schirmer K: Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 2006, 224:163-183. 13. Kramer NI, Hermens JLM, Schirmer K: The influence of modes of action and physicochemical properties of chemicals on the correlation between in vitro and acute fish toxicity data. Toxicol In Vitro 2009, 23:1372-1379. 14. Scholz S, Sela E, Blaha L, Braunbeck T, Galay-Burgos M, Garcı´a-Franco M, Guinea J, Klu¨ver N, Schirmer K, Tanneberger K et al.: A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment. Regul Toxicol Pharmacol 2013, 67:506-530. www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 67
15. Bury NR, Schnell S, Hogstrand C: Gill cell culture systems as models for aquatic environmental monitoring. J Exp Biol 2014, 217:639-650. 16. Dayeh VR, Schirmer K, Bols NC: Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent. Water Res 2002, 36:3727-3738. 17. Castan˜o A, Bols N, Braunbeck T, Dierickx P, Halder M, Isomaa B, Kawahara K, Lee LEJ, Mothersill C, Pa¨rt P et al.: The use of fish cells in ecotoxicology. The report and recommendations of ECVAM Workshop 47. Altern Lab Anim ATLA 2003, 31:317-351. 18. Segner H: Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. Altern Lab Anim ATLA 2004, 32:375-382. 19. Bols NC, Dayeh VR, Lee LEJ, Schirmer K: Chapter 2 use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology [Internet]. Biochemistry and Molecular Biology of Fishes. Elsevier; 2005:: 43-84. 20. Dayeh VR, Bols NC, Tanneberger K, Schirmer K, Lee LEJ: The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD’s fish toxicity testing framework No. 171. In Curr Protoc Toxicol Editor. Board. Edited by Maines M. 2013. Chapter 1: Unit 1.5. 21. Giaever I, Keese CR: Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A 1984, 81:3761-3764. 22. Jiang WG (Ed): Electric Cell-substrate Impedance Sensing and Cancer Metastasis. Springer; 2012. 23. van der Schalie WH, James RR, Gargan TP: Selection of a battery of rapid toxicity sensors for drinking water evaluation. Biosens Bioelectron 2006, 22:18-27. 24. Widder MW, Trader D, Brennan L, Shedd TR, Gargan TP II, van der Schalie WH, Salazar N, Vallejo YR: An Environmental Sentinel Biomonitor System for Drinking Water Protection [Internet]. DTIC Document; 2008. 25. Curtis TM, Widder MW, Brennan LM, Schwager SJ, van der Schalie WH, Fey J, Salazar N: A portable cell-based impedance sensor for toxicity testing of drinking water. Lab Chip 2009, 9:2176-2183. 26. Brennan LM, Widder MW, Lee LEJ, van der Schalie WH: Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells. Toxicol Vitro Int J Publ Assoc BIBRA 2012, 26:736-745. 27. Widder MW, Brennan LM, Hanft EA, Schrock ME, James RR, van der Schalie WH: Evaluation and refinement of a field-portable drinking water toxicity sensor utilizing electric cell-substrate impedance sensing and a fluidic biochip: evaluation and refinement of a field-portable water toxicity sensor. J Appl Toxicol 2015, 35:701-708. This paper accounted some of the latest developments of the impedance-based biosensor developed by the US Army. The current prototype is a portable device using RTgill-W1 cells with optimised procedural steps, and reflects the essence of the decade-long pursuit of this line of biosensor. 28. Brennan LM, Widder MW, McAleer MK, Mayo MW, Greis AP, van der Schalie WH: Preparation and testing of impedance-based fluidic biochips with RTgill-W1 cells for rapid evaluation of drinking water samples for toxicity. J Vis Exp 2016 http://dx.doi. org/10.3791/53555. This work contains a step-by-step protocol of using the impedancebased biosensor developed by the US Army. The video and guide could be very useful for end users and developers of similar biosensors. 29. Pan T, Li H, Khare S, Huang B, Yu Huang D, Zhang W, Gabos S: High-throughput screening assay for the environmental water samples using cellular response profiles. Ecotoxicol Environ Saf 2015, 114:134-142. The study tested real water samples with xCELLigence using a variety of mammalian cell lines. It contains useful information of data evaluation and about environmental water sample monitoring on an impedance-based test platform. 30. Bohrn U, Mucha A, Werner CF, Trattner B, Ba¨cker M, Krumbe C, Schienle M, Stu¨tz E, Schmitt-Landsiedel D, Fleischer M et al.: www.sciencedirect.com
A critical comparison of cell-based sensor systems for the detection of Cr(VI) in aquatic environment. Sens Actuators B Chem 2013, 182:58-65. 31. Rakers S, Imse F, Gebert M: Real-time cell analysis: sensitivity of different vertebrate cell cultures to copper sulfate measured by xCELLigenceJ. Ecotoxicology 2014, 23:15821591. This study tested xCELLigence with mammalian and fish cell lines, and compared the data with conventional assays. It provides a good overview of how toxicity was manifested among different cell lines and reflected among different assays. 32. Kubisch R, Bohrn U, Fleischer M, Stu¨tz E: Cell-based sensor system using L6 cells for broad band continuous pollutant monitoring in aquatic environments. Sensors 2012, 12:3370-3393. 33. Liu F, Chen T, Zhang X, Li F, Voiculescu I: Study of Long Term Viability of Endothelial Cells on Biochip for Rapid and Reliable Water Toxicity Measurements. ASME; 2013:. V001T02A002V001T02A002. 34. Liu F, Li F, Nordin AN, Voiculescu I: A novel cell-based hybrid acoustic wave biosensor with impedimetric sensing capabilities. Sensors 2013, 13:3039-3055. 35. Liu F, Nordin AN, Li F, Voiculescu I: A lab-on-chip cell-based biosensor for label-free sensing of water toxicants. Lab Chip 2014, 14:1270-1280. This study showed that the combination of two biosensing mechanisms (impedance and quartz crystal microbalance resonator) resulted in increased accuracy of detection. It is a typical example of integrating multiparametric biosensors for enhanced performance. 36. Chaplen FW, Upson RH, Mcfadden PN, Kolodziej W: Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens. Pigment Cell Res 2002, 15:19-26. This is a pioneer study of chromatophore-based biosensors. Its development led to the successful application of a patent, a chain of events that were not often observed in biosensor development for water toxicity. Encouraging such initiatives and routes would help promote the adoptation and field application of biosensors for water monitoring. 37. Dierksen KP, Mojovic L, Caldwell BA, Preston RR, Upson R, Lawrence J, McFadden PN, Trempy JE: Responses of fish chromatophore-based cytosensor to a broad range of biological agents. J Appl Toxicol JAT 2004, 24:363-369. LV, Jovanovic GN: Development of a microbiosensor 38. Mojovic based on fish chromatophores immobilized on ferromagnetic gelatin beads. Food Technol Biotechnol 2005, 43:1-7. 39. Plant TK, Chaplen FW, Jovanovic G, Kolodziej W, Trempy JE, Willard C, Liburdy JA, Pence DV, Paul BK: Sensitive-cell-based Fish Chromatophore Biosensor. Edited by Mahadevan-Jansen A, Sowa MG, Puppels GJ, Gryczynski Z, Vo-Dinh T, Lakowicz JR.2004:265. 40. Sharma V, Narayanan A, Rengachari T, Temes GC, Chaplen F, Moon U-K: A low-cost, portable generic biotoxicity assay for environmental monitoring applications. Biosens Bioelectron 2005, 20:2218-2227. 41. Dukovcic SR, Hutchison JR, Trempy JE: Potential of the melanophore pigment response for detection of bacterial toxicity. Appl Environ Microbiol 2010, 76:8243-8246. 42. Dukovcic SR, Hutchison JR, Trempy JE: Conservation of the chromatophore pigment response. J Appl Toxicol 2010, 30:574581. 43. Iuga A, Lerner E, Shedd TR, van der Schalie WH: Rapid responses of a melanophore cell line to chemical contaminants in water. J Appl Toxicol 2009, 29:346-349. 44. Polak ME, Rawson DM, Haggett BGD: Redox mediated biosensors incorporating cultured fish cells for toxicity assessment. Biosens Bioelectron 1996, 11:1253-1257. 45. Wang S, Ramachandran A, Ja S-J: Integrated microring resonator biosensors for monitoring cell growth and detection of toxic chemicals in water. Biosens Bioelectron 2009, 24:3061-3066. Current Opinion in Biotechnology 2017, 45:59–68
68 Environmental biotechnology
46. Cevenini L, Calabretta MM, Tarantino G, Michelini E, Roda A: Smartphone-interfaced 3D printed toxicity biosensor integrating bioluminescent “sentinel cells”. Sens Actuators B Chem 2016, 225:249-257. 47. Bols NC, Barlian A, Chirino-Trejo M, Caldwell SJ, Goegan P, Lee LEJ: Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. J Fish Dis 1994, 17:601-611. 48. Dayeh VR, Grominsky S, DeWitte-Orr SJ, Sotornik D, Yeung CR, Lee LEJ, Lynn DH, Bols NC: Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena. Res Microbiol 2005, 156:93-103.
53. Schirmer K, Dixon D, Greenberg B, Bols N: Ability of 16 priority PAHs to be directly cytotoxic to a cell line from the rainbow trout gill. Toxicology 1998, 127:129-141. 54. Dayeh VR, Schirmer K, Bols NC: Ammonia-containing industrial effluents, lethal to rainbow trout, induce vacuolisation and Neutral Red uptake in the rainbow trout gill cell line, RTgill-W1. Altern Lab Anim ATLA 2009, 37:77-87. 55. Nano-Tera Swiss National Science Foundation: Envirobot. URL: https://wp.unil.ch/envirobot/. [Accessed 31 October 2016]. 56. Research consortium Envirobot: Envirobot. URL: http://www. nano-tera.ch/projects/345.php#desc. [Accessed 31 October 2016].
49. Lee LE, Dayeh VR, Schirmer K, Bols NC: Fish cell lines as rapid and inexpensive screening and supplemental tools for whole effluent testing. Integr Environ Assess Manag 2008, 4:372.
57. van der Meer JR: Towards improved biomonitoring tools for an intensified sustainable multi-use environment. Microb Biotechnol 2016, 9:658-665.
50. Glawdel T, Elbuken C, Lee LEJ, Ren CL: Microfluidic system with integrated electroosmotic pumps, concentration gradient generator and fish cell line (RTgill-W1)—towards water toxicity testing. Lab Chip 2009, 9:3243.
58. Chaplen F, Gerwick W, Jovanovic G, Kolodziej W, Liburdy JA, McFadden PN, Paul BK, Plant TK, Trempy JE, Willard C: Methods for detecting bioactive compounds. 2005, Patent No. US6913877 B1.
51. Lee LEJ, Dayeh VR, Schirmer K, Bols NC: Applications and potential uses of fish gill cell lines: examples with RTgill-W1. Vitro Cell Dev Biol—Anim 2009, 45:127-134.
59. Widder MW, Brennan LM, Trader D, Shedd TR, van der Schalie WH: Environmental Sentinel Biomonitor System (ESB). 2012, Patent Application No. US 13/156,826.
52. Yue Y, Behra R, Sigg L, Schirmer K: Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium. Nanotoxicology 2016, 10:1075-1083.
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com
ScienceDirect Cell culture-based biosensing techniques for detecting toxicity in water Lu Tan1 and Kristin Schirmer1,2,3 The significant increase of contaminants entering fresh water bodies calls for the development of rapid and reliable methods to monitor the aquatic environment and to detect water toxicity. Cell culture-based biosensing techniques utilise the overall cytotoxic response to external stimuli, mediated by a transduced signal, to specify the toxicity of aqueous samples. These biosensing techniques can effectively indicate water toxicity for human safety and aquatic organism health. In this review we account for the recent developments of the mainstream cell culture-based biosensing techniques for water quality evaluation, discuss their key features, potentials and limitations, and outline the future prospects of their development. Addresses 1 Department of Environmental Toxicology, Eawag, Swiss Federal Institute of Aquatic Science and Technology, U¨berlandstrasse 133, 8600 Du¨bendorf, Switzerland 2 School of Architecture, Civil and Environmental Engineering (ENAC), EPF Lausanne, 1015 Lausanne, Switzerland 3 Institute of Biogeochemistry and Pollutant Dynamics, ETH Zu¨rich, 8092 Zu¨rich, Switzerland Corresponding author: Schirmer, Kristin ([email protected])
Current Opinion in Biotechnology 2017, 45:59–68 This review comes from a themed issue on Environmental biotechnology Edited by Jan Roelof Van Der Meer and Man Bock Gu For a complete overview see the Issue and the Editorial Available online 27th January 2017 http://dx.doi.org/10.1016/j.copbio.2016.11.026 0958-1669/ã 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).
Introduction Today in both developing and industrialised countries, an increasing number of contaminants, pertaining to 300 million tons of industrial chemicals and 145 million tons of fertilisers and pesticides produced worldwide annually, are entering the water bodies [1]. They are impairing ecosystems, and in turn, threatening the water security of the world’s population [2,3]. In the US, about half of the assessed stream miles and lake acres, and one third of the assessed bay and estuarine square miles are classified as polluted; in China, the majority of rivers and lakes, and all of its coastal waters are polluted [4]. In the European Union, only a fraction of the www.sciencedirect.com
>100 000 EINECS (European INventory of Existing Commercial chemical Substances) registered chemicals have their assessment reports available in the OECD (Organisation for Economic Co-operation and Development) Existing Chemicals Database [5]. The effects of most chemicals on the ecosystem are largely unknown. Increasing initiatives and legislative actions for water pollution control have been adopted [6]; the Water Framework Directive (WFD) has been established to govern the European water policy, which places aquatic ecology at the basis of water management decisions [7,8]. The demand for restoring the water status calls for rapid, reliable and economic methods for water monitoring and chemical toxicological impact identification and assessment [9]. The fish acute toxicity test (OECD test guideline 203) is the most widely applied assay for aquatic environmental risk assessment, where 42–60 fish are exposed for 96 hours and death is used as end point. The latest available data showed that, in the EU alone, 179 083 fish were used in regulatory toxicological and safety evaluations in 2011 [10], whilst an estimated three million fish are used in North America for effluent testing each year [11]. Besides being technically challenging, time-consuming and costly, fish tests also raise ethical concerns of animal welfare; thus there is a strong demand for replacement, reduction and refinement approaches [12,13,14,15]. The past years have seen many efforts to develop alternatives for animal testing, and the most extensively explored are the in vitro cell models [11,16,17,18,19]. A variety of fish cell-based methods have been developed in testing of chemical and whole water effluents, and shown to be effective, reproducible and economic [12]. Recently, close sensitivity and correlation between rainbow trout gill cell (RTgill-W1) cytotoxicity and acute fish toxicity have been demonstrated [11], indicating the predictive power of cell models and their promising application as alternatives for animal experimentation. It is generally accepted that the replacement of whole animal tests by a single alternative method is difficult, but the use of alternatives to significantly reduce animal testing is desired and possible [14]. Biosensors, incorporating cell-mediated biological recognition systems and signal transducing elements, offer even more advantages of rapid response, portability, field applicability, being real-time and cost-effective, compared to the conventional cell-based bioassays [19,20], and thus are excellent choices for water status monitoring Current Opinion in Biotechnology 2017, 45:59–68
60 Environmental biotechnology
and chemical toxicity detection. Most ‘cell-based’ biosensors refer to microbes, which are not within the scope of this review. The term ‘cell’ in the following context all refers to vertebrate cells originating from different tissues. Biosensor developments for water toxicity detection often originate from either human safety (drinking water quality) or ecotoxicological concerns (water quality for biota and the aquatic environment). However, as many cell-based biosensors can be used for water quality evaluation of both human and ecosystem health, boundaries for their applications are rather blurry. Therefore, in the techniques reviewed here, we do not distinguish whether a method was originally developed for human safety or
ecotoxicology. Figure 1 summarises the basics of cell culture-based biosensors. In the review we will highlight the recent major developments of cell-based biosensing methods for water toxicity, and discuss their features, potentials, limitations and further developments.
Cell-based biosensing techniques Electric cell-substrate impedance sensing (ECIS)
The most vibrant research activity in cell-based biosensing for water assessment builds on the Electric Cellsubstrate Impedance Sensing [21] (ECIS) technology. ECIS utilises cell culture medium as electrolyte with cells grown on an electrode-embedded culturing surface.
Figure 1
Detectable contaminants
Contaminants in water
Cells
Industrial chemicals
Heavy metals
Pharmaceuticals & pesticides
Pathogens & their toxins
PAHs
Chem. mixtures in fresh- & wastewater
Toxicity manifestation
Etc.
Applications Etc. Detachment Morphology change
Death
Pigment translocation
On-site chemical impact assessment
Ecosystem conservation
Signal transduction and toxicity indication Water monitoring
Contaminated
or
Not Contaminated
Early warning system
Current Opinion in Biotechnology
Cell-based biosensors for detecting toxicity in water. Known or unknown contaminants in water elicit various toxic effects in cells, which can be manifested as cell detachment, morphology change, cell death, and in case of chromatophores, the translocation of pigments. These cytotoxic signals can be transduced in biosensors, by, for example cell impedance or pigment tracking, to an integrated receiving end to indicate the toxicity. Cell-based biosensors are able to detect a broad spectrum of substances and can be applied in many different areas of toxicity detection in water. Some of the applications may share some common aims (overlapping circles).
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 61
By applying a very low, non-invasive alternating current, the cell electric impedance can be measured to indicate the cell status including growth, migration, morphology, cell-to-matrix and cell-to-cell interactions [22]. Research using ECIS technology to evaluate water quality has been consistently led by the US Army Center for Environmental Health Research for the purpose of developing a fielduse device for Army personnel deployed around the world to rapidly test drinking water for possible chemical contaminations [23,24]. The decade-long development route [23,24,25,26,27,28] (Table 1, ECIS) started with an initial testing of a dozen of toxic industrial chemicals using 10 different sensors, with results putting ECIS among the top performing technology [23]. The openwell ECIS platform was combined with an enzyme-based acetylcholinesterase inhibition sensor, to form the Environmental Sentinel Biomonitor (ESB) system [24]. The ECIS platform was further modified and refined to a portable automated device incorporating a self-contained disposable media delivery system [25]. By this point, cells used were mammalian cells that required tightly controlled maintenance condition. This new system enabled the long-term keeping of the mammalian cells (up to 16 weeks of responsiveness) through the automated media delivery system, which was a significant step forward. Further milestone development adopted the rainbow trout gill cells (RTgill-W1) that showed comparable performance as mammalian cells, and significantly more robustness and simple maintenance with a tested 39 weeks of responsiveness [26]. This study fulfilled the prospect of using the system as a portable, low-cost, lowmaintenance biosensor. Follow-up researches [27,28] refined the procedural steps and added a hand-held reader displaying ‘contaminated’ or ‘not contaminated’ result for non-expert users, readying the device for military and municipal field-use. Figure 2a,b show the RTgill-W1 cells and the ECIS biochip. Although the systematic development procedure solved many technical issues, limitations inherent to the cell line, such as the insensitivity to certain classes of stimuli could not be overcome using the ECIS biosensor alone. Other impedance-based methods
Several commercial systems based on cell impedance, such as xCELLigence (ACEA Biosciences, USA) and Bionas (Bionas, Germany), often applied in pharmaceutical research, have also been used in assessing toxicity in water. xCELLigence (Table 1, xCELLigence) measures cell impedance represented as Cell Index (CI) that reflects cell growth, death, adhesion and morphology [29]. The larger the CI value, the more viable cells are attached to the system. Perturbation by external stimuli can be detected via the change of CI values. Eight mammalian cell lines and a rainbow trout skin derived cell line were tested using xCELLigence in various studies [29,30,31]. Although there were variations comparing the impedance method with viability www.sciencedirect.com
assays such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), and also differences between cell lines within the same assay, the xCELLigence system was generally effective in reacting to purposely spiked toxicants in water [31] as well as to real water samples [29]. Bionas (Table 1, Bionas) is a multiparametric system integrating three parameter read-outs: cell impedance, acidification (metabolism) and oxygen consumption (respiration); five mammalian cell lines were tested with Bionas [30,32]. The integrative use of multiple parameters, particularly oxygen consumption, enabled more rapid detection of toxicants [30]. The system was suitable for detecting a broad range of substances including heavy metals, pharmaceuticals, neurotoxins and contaminants in wastewater [32]. Another multiparametric biosensor (Table 1, ECIS/QCM) with impedance and mass-sensing through a Quartz Crystal Microbalance (QCM) resonator was developed and shown to be effective in fast detection of various chemicals with increased accuracy [33,34,35]. Chromatophore-based approaches
Chromatophores are neurone-like pigmented cells found in amphibians, fish and reptiles, and are able to respond to stimuli with changing pigment distribution. The pigment aggregation and dispersion are mainly mediated by Gprotein linked signal transduction involving phosphorylation events and motor protein mobilisation to translocate the pigment granules [36]. In chromatophore-based water toxicity biosensing (Table 2, Chromatophore-based approaches), the most commonly applied chromatophores originated from Siamese fighting fish [36,37,38,39,40], and also from Chinook salmon [41,42], Nile tilapia [23] and African clawed frog [43]. These cells are shown to respond to live pathogenic microbes or microbial toxins and a wide range of chemicals including heavy metals, organophosphate pesticides and polynuclear aromatic hydrocarbons (PAHs) [23,36,40]. Although the fish species inhabit different ecological niches, their responses to external stimuli are highly conserved [41,42], suggesting that similar cellular mechanisms govern the change of pigment distribution. An example of chromatophore pigment relocation is shown in Figure 2c. This technique was pioneered by integrating light source, camera and lens, monitor and keyboard and an image processing software into a portable device [36]. Further chromatophore-based biosensing studies adopted similar setup components [23,37]. Quantification methods have been a key issue in interpreting chromatophore test results, mainly caused by the heterogeneity of fast- and slowresponding cells, the variation in number and density of chromatophore samples, and the ambiguity of subtle changes [36,38,40]. Modelling and refinement in quantitative analysis are often necessary; commonly applied methods quantify changes of cell area covered by pigment [23,37,38] using photographic recording of cells upon a fixed field of view containing the same number Current Opinion in Biotechnology 2017, 45:59–68
62 Environmental biotechnology
Table 1 Impedance-based biosensing techniques for water toxicity detection ECIS Cell
Cell type
Origin
Biosensing mechanism
Tested substances
Test time a
Longevity
Realtime
Ref.
BPAEC
Mammalian
x
[23]
1 hour
30 days
x
[24]
BPAEC
Mammalian
1 hour
9 days
x
[25]
BLMVEC
Mammalian
Bovine lung microvessel endothelial cells
1 hour
16 weeks
x
[25]
RTgill-W1
Fish
1 hour
39 weeks
x
[26]
BLMVEC
Mammalian
16 weeks
x
[26]
Fish
18 chemicals including aldicarb, ammonia, paraquat, and so on 18 chemicals including aldicarb, ammonia, paraquat, and so on
1 hour
RTgill-W1
1 hour
N. M.
x
[27]
RTgill-W1
Fish
Rainbow trout (Oncorhynchus mykiss) gill cells Bovine lung microvessel endothelial cells Rainbow trout (Oncorhynchus mykiss) gill cells Rainbow trout (Oncorhynchus mykiss) gill cells
12 chemicals incl. aldicarb, ammonia, paraquat, and so on 12 chemicals incl. aldicarb, ammonia, paraquat, and so on Five chemicals incl. phenol, potassium cyanide, pentachlorophenate, and so on Five chemicals incl. phenol, potassium cyanide, pentachlorophenate, and so on 18 chemicals including aldicarb, ammonia, paraquat, and so on
N. M. b
Mammalian
Changes in cell-substrate impedance after perturbation by chemicals
1 hour
BLMVEC
Bovine pulmonary artery endothelial cells Bovine lung microvessel endothelial cells Bovine pulmonary artery endothelial cells
13 chemicals including aldicarb, ammonia, paraquat, and so on
1 hour
N. M.
x
[28]
Longevity
Realtime
Ref.
xCELLigence Cell
Cell type
Origin
Biosensing mechanism
V79
Mammalian
NIH-3T3
Mammalian
Impedance measured as cell index
RAsd CEsd
Mammalian Mammalian
OMYsd
Fish
A549
Mammalian
ACHN
Mammalian
HepG2
Mammalian
SK-N-SH
Mammalian
Chinese hamster lung cells Mouse embryo fibroblast cells Rat skin derived cells Human skin derived cells Rainbow trout (Oncorhynchus mykiss) skin derived cells Human lung epithelial cells Human kidney derived cells Human hepatoblastoma cells Human neuroblastoma cells
Tested substances
Test time
N. M.
x
[30]
Copper sulfate pentahydrate
Several to 24 hours 24 hours
N. M.
x
[31]
Copper sulfate pentahydrate Copper sulfate pentahydrate
24 hours 24 hours
N. M. N. M.
x x
[31] [31]
Copper sulfate pentahydrate
24 hours
N. M.
x
[31]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Samples of fresh water
128 hours
N. M.
x
[29]
Chromium
Bionas Cell
Cell type
Origin
V79
Mammalian
HT-29
Mammalian
L6
Mammalian
Chinese hamster lung cells Human colon adenocarcinoma Rat skeletal muscle
HepG2
Mammalian
V79
Mammalian
Biosensing mechanism Multiparametric: changes in impedance, pH and oxygen consumption
Human hepatoblastoma cells Chinese hamster lung cells
Current Opinion in Biotechnology 2017, 45:59–68
Tested substances Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Heavy metals, pharmaceuticals, neurotoxins, wastewater Chromium
Test time
Longevity
Realtime
Ref.
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
24 hours
N. M.
x
[32]
Several to 24 hours
N. M.
x
[30]
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 63
Table 1 (Continued ) ECIS/QCM Cell
Cell type
BAEC
Mammalian
BAEC
Mammalian
BAEC
Mammalian
Origin Bovine aortic endothelial cells Bovine aortic endothelial cells Bovine aortic endothelial cells
Biosensing mechanism Multiparametric: changes in impedance and resonance frequency through mass sensor
Tested substances
Test time
Longevity
Realtime
Ref.
Ammonia
1 hour
34 days
x
[33]
Cell behaviour characterisation Ammonia, nicotine and aldicarb
Several days
6 weeks
x
[34]
Up to 1 hour
30 days
x
[35]
References in each category are listed by time of publication (most recent at the end). Is taken from method description if available, or from information in diagrams if not specified in method. Response time is commonly within or close to test time. b N. M. = not mentioned. This information may be available in other studies that tested the longevity for the same cell line under similar conditions. a
of cells. That chromatophores are primary cells and unable to continuously propagate in culture, adds to the complexity of data interpretation and reproducibility, and limits their application for routine water toxicity evaluation.
Besides the impedance- and chromatophore-based approaches, several other techniques were also tested in water toxicity detection. These researches utilised distinct biosensing mechanisms [23,44,45,46] (details in Table 2, Other approaches) but were more fragmented,
Figure 2
(a)
(b)
250 µm
50 µm (c)
Current Opinion in Biotechnology
Examples of cells in cell-based biosensors for water toxicity detection. (a) Live cell staining of the RTgill-W1 cells, where nuclei (blue), cell membrane (green), and lysosome (magenta) are visible. (b) With permission from Ref. [26], ECIS biochip with RTgill-W1 cells. (c) With permission from Ref. [40], aggregation of pigment (right) in chromatophores (untreated, left) of Siamese fighting fish after treatment with norepinephrine (1 mg/ml). www.sciencedirect.com
Current Opinion in Biotechnology 2017, 45:59–68
64 Environmental biotechnology
Table 2 Chromatophore-based biosensing techniques and other approaches for water toxicity detection Chromatophore-based approaches Cell
Cell type
Origin
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Siamese fighting fish (Betta splendens)
Chromatophores
Fish
Nile tilapia (Oreochromis niloticus)
Melanophores
Amphibian
Melanophores
Fish
Melanophores
Fish
Melanophores from African clawed frog (Xenopus laevis) Chinook salmon (Oncorhynchus tshawytscha) Chinook salmon (Oncorhynchus tshawytscha)
Biosensing mechanism Translocation of cellular pigments upon perturbation of cellular processes Translocation of cellular pigments upon perturbation of cellular processes Changes in the total area of colour in the cells Changes in pigment covered area in cells immobilised to beads Changes in light transmitted through the cell area of the chromatophores Changes in chromatophore distribution Changes in light absorption by the cells after chemical exposure Changes in cell pigments Changes in cell pigments
Test time a
Longevity
Ref.
Bacterial toxins and chemicals such as PAHs
1 hour
3 months
[36]
Various cellular effectors and bacterial toxins
1 hour
N. M.
Bacterial toxins
20 min
13 weeks
[39]
Clonidine
6 min
2–4 weeks
[38]
Clonidine and cirazoline
5 min
Up to 1 month
[40]
12 chemicals incl. aldicarb, ammonia, paraquat, and so on 12 chemicals incl. aldicarb, ammonia, paraquat, and so on Pathogens
1 hour
N. M.
[23]
1 hour
N. M.
[43]
20 min
N. M.
[41]
Environmental toxicants incl. mercuric chloride and sodium arsenite
1 hour
3 weeks
[42]
Tested substances
b
[37]
Other approaches Cell
Cell type
Origin
Biosensing mechanism
Tested substances
Test time
Longevity
Ref.
Epithelioma papulosum cyprini derived from a skin tumour of carp (Cyprinus carpio) Human hepatoblastoma cells
Redox mediated currents generated by metabolising cells when perturbed by chemicals Changes of fluorophore-labelled LDL-update in cells after contaminant exposure Shift in refractive index at the microring resonator surface caused by chemical toxicity Changes in bioluminescence
p-Benzoquinone; 2,6dimethylbenzoquinone
1 hour
N. M.
[44]
12 chemicals incl. aldicarb, ammonia, paraquat, and so on
2 hours
N. M.
[23]
Sodium pentachlorophenate and aldicarb
1 hour
2 days
[45]
Aqueous samples from daily life
30 min
2–3 days
[46]
EPC
Fish
Hep G2
Mammalian
MS1
Mammalian
Mile Sven 1 of pancreas from mouse
Hek293T
Mammalian
Human embryonic kidney cells, engineered, bioluminescent
References in each category are listed by time of publication (most recent at the end). Is taken from method description if available, or from information in diagrams if not specified in method. Response time is commonly within or close to test time. b N. M. = not mentioned. This information may be available in other studies that tested the longevity for the same cell line under similar conditions. a
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 65
involving few precedence or follow-up of development or application.
Key features of cell-based biosensing techniques Choices of cells, longevity and stability
The majority of cell lines used in the mainstream impedance-based water toxicity biosensors are mammalian cells, with the consideration that mammalian cells would better address the toxic responses relevant to human health and safety. However, both mammalian and fish cells were able to respond to contaminants within toxic ranges, and were comparable in relative terms in all available biosensing studies [26,31]. This is largely caused by the fact that cell-based biosensors detect the overall cytotoxic effects, which is essentially a response of basal toxicity common to all cells. However, as the fledgling technology developed to fit practicality for realistic applications, the need for rigorous maintenance and the limited longevity and stability of mammalian cells became a major hurdle. The earlier ECIS-based systems used the relatively robust mammalian cells from the bovine pulmonary tissues [23,24,25,26,33,34,35] which were shown to stay viable and responsive for one [33] to three months [25]. Nevertheless, these cells require medium replenishment several times a week and maintenance at 37 C and 5% of CO2, which significantly reduces their applicability in a portable field-device. The later development of ECIS adopted the rainbow trout gill cells (RTgill-W1) [47], which is a continuous cell line and can survive across a wide range of temperature (4–24 C) with optimal growth condition at 20 C and no requirement on CO2 [48]. This cell line is one of the most extensively researched and applied models in the context of alternatives to fish tests and water toxicity [11,48,49,50,51,52]. Its cytotoxicity is closely correlated to fish data; it can be used as a predictor for in vivo acute toxicity [11]. RTgill-W1 can be exposed in serum-free conditions to eliminate interference of serum proteins with toxicants [11]. These cells do not require media change at low-temperature storage condition, and have been tested with toxicant responses after about nine months of storage [26]. These characteristics clearly advantage fish cells over mammalian cells for biosensing applications in water. In the chromatophore-based approaches, cells are mainly isolated from fish and have been shown to have an active life of several weeks [38,41] up to three months [36,39]. Unlike the continuous cell lines applied in impedance-based approaches, chromatophores are primary cells and have the inherent limitation in batch-to-batch variability and reproducibility. Detectable substances
Cells used in impedance-based biosensing detect a wide range of toxic substances including heavy metals, industrial chemicals, pharmaceuticals, and chemical mixtures www.sciencedirect.com
in freshwater and wastewater [23,24,25,26,27,28,29, 30,31,32,34,35]. Furthermore, fish cells have been used in water evaluation in conventional bioassays with larger collection of chemicals and environmental samples, including PAHs [53], industrial effluents [16], ammonia [54], diverse organic chemicals [11], and nanoparticles [52]. Such substances and samples should also be able to elicit positive responses on the cell-based impedance biosensing. However, inherent to lacking of receptors in certain tissues, not all cells are sensitive to all types of chemical classes. For example, the neurotoxic chemicals permethrin and lindane were significantly less acutely toxic to RTgill-W1 cells than to fish [11]. Chromatophores have been shown to be sensitive to toxic industrial chemicals and environmental toxicants [23,42,43], live microbial pathogens and bacterial toxins [36,37,39,41]. Response time and real-time testing
Rapid response time is an important criterion for aquatic toxicity biosensing. In the series of ECIS biosensing development, cells responded to many of the tested chemicals within one hour of exposure [23,24,25,26, 27,28]. Monitoring of chemical exposure on the commercial devices xCELLigence and Bionas were usually set to be several to 24 hours [30,31,32], and the freshwater samples to over 100 hours [29]. The chromatophorebased approaches could also detect chemical toxicity in acute exposure of about one hour [42,43]. It needs to be noted that response time is largely determined by the test doses. The criteria in the ECIS studies were set to be between the MEG (Military Exposure Guidelines) and HLC (Human Lethal Concentration) [23,26,27], where the HLC could be one to five orders-of-magnitude higher than the MEG [26]. Within one hour of exposure, toxicant dose was usually much higher than the MEG, and closer to HLC [26]. In the conventional bioassays, however, cells were usually exposed for 24 hours with a range of doses [11]. The higher the toxicant dose, the more rapid the cellular responses are likely to be. Freshwater samples could be much less toxic than spiked and wastewater samples and thus require longer exposure time; for example, a study using freshwater samples carried out the monitoring for several days [29]. Therefore, the term ‘rapid response’ needs always to be interpreted within a context in relation to chemical dosage. In field applications, a ‘rapid response’ would be likely in a chemical spill accident or wastewater, and less likely in monitoring of eventless freshwater bodies in little polluted regions. Real-time monitoring is an advantage of impedancebased biosensing technology. The time-dependent changes of impedance can be recorded throughout chemical exposure. This enables continuous observation of the system, and termination of the tests at any time (i.e. when a positive response is visible). This offers particular advantages in testing samples with unknown substances. Current Opinion in Biotechnology 2017, 45:59–68
66 Environmental biotechnology
Combined and multiparametric biosensors
The testing on overall cytotoxicity can be extended to other, i.e. more specific assays. Two trends dominate the development of complementing systems. One is the combined use of other biosensors, where a battery of multiple sensors provides an even wider range of chemical detection [23]. The other is the integration of several biosensing techniques into one multiparametric device [24,30,32,33,34,35]. The enzyme-based acetylcholinesterase inhibition test was added to ECIS in the ESB system [24,27,28]. The Bionas utilised three parameters including impedance, pH and respiration, and was shown to enable much faster detection of chromium with the respiration assay [30,32]. Moreover, the weight-sensing QCM technique was integrated with ECIS for enhanced sensitivity and accuracy in chemical detection [33,34,35].
Conclusions Important developments took place in recent years in cell-based biosensing techniques for water quality assessment. In future developments, continued exploration in impedance-based approaches using fish cell models, such as RTgill-W1 cells, will boost the field-applicability of cell-based biosensors and bridge the gap in the variety of tested chemicals/samples between conventional bioassays and biosensors. This will improve our understanding of the biosensing mechanism and efficacy, and the approach will also add to the plethora of means in reducing animal usage and finding alternatives to animal testing. Chromatophores offer the distinct direction of rapid biosensing based on optic transduction, but its further development largely depends on overcoming the limitations associated with primary cell cultures, heterogeneity and reproducibility. Since many non-chromatophore, continuous fish cell lines were spontaneously immortalised, the possibilities to immortalise chromatophores and stabilise their variability merit further investigation. Since a single type of cells is not able to respond to all kinds of contaminants, based on the application purpose, combinatorial or multiparametric biosensing can complement the cell-based biosensors and will be a trending focus in biosensor development. The Envirobot [55,56,57], a compartmented autonomous robotic system incorporating various biological sensors based on vertebrate cells, daphnia, amphibian oocytes, microorganism, etc., is a leading example of large-scale multiparametric biosensing for water quality. Although a number of cell culture-based biosensors have been integrated, field testing and actual applications are still limited. The ability to transform a scientific research project to application is the key. This transformation is demanded by the urgent need for effective water monitoring methods, and has seen some endeavours in obtaining product development protection through patent applications [58,59]. In the future, actively bridging the legislative requirements and water initiatives during sensor development, as well as intensified Current Opinion in Biotechnology 2017, 45:59–68
exploration of proprietary rights and commercialisation routes, will significantly help implementing the real-life usage of water toxicity biosensors in various applications.
Acknowledgement The authors acknowledge the financial support from Swiss National Science Foundation Nano-Tera, project Envirobot, grant 20NA21-143082.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Schwarzenbach RP: The challenge of micropollutants in aquatic systems. Science 2006, 313:1072-1077.
2.
Shannon MA, Bohn PW, Elimelech M, Georgiadis JG, Marin˜as BJ, Mayes AM: Science and technology for water purification in the coming decades. Nature 2008, 452:301-310.
3.
Vo¨ro¨smarty CJ, McIntyre PB, Gessner MO, Dudgeon D, Prusevich A, Green P, Glidden S, Bunn SE, Sullivan CA, Liermann CR et al.: Global threats to human water security and river biodiversity. Nature 2010, 467:555-561.
4.
Eltzov E, Marks RS: Whole-cell aquatic biosensors. Anal Bioanal Chem 2011, 400:895-913.
5.
OECD: Status of Chemicals in the Assessment Process. OECD; 2016 http://webnet.oecd.org/hpv/ui/OverallStatus.aspx.
6.
Farre´ M, Barcelo´ D: Biosensors for aquatic toxicology evaluation. In Biosensors for Environmental Monitoring of Aquatic Systems. Edited by Barcelo´ D, Hansen P-D. Berlin, Heidelberg: Springer; 2009:115-160.
7.
Namour P, Lepot M, Jaffrezic-Renault N: Recent trends in monitoring of European Water Framework Directive priority substances using micro-sensors: a 2007–2009 review. Sensors 2010, 10:7947-7978.
8.
Hering D, Borja A, Carstensen J, Carvalho L, Elliott M, Feld CK, Heiskanen A-S, Johnson RK, Moe J, Pont D: The European Water Framework Directive at the age of 10: a critical review of the achievements with recommendations for the future. Sci Total Environ 2010, 408:4007-4019.
9.
Lagarde F, Jaffrezic-Renault N: Cell-based electrochemical biosensors for water quality assessment. Anal Bioanal Chem 2011, 400:947-964.
10. EU: Seventh Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member States of the European Union. European Commission; 2013. 11. Tanneberger K, Kno¨bel M, Busser FJM, Sinnige TL, Hermens JLM, Schirmer K: Predicting fish acute toxicity using a fish gill cell line-based toxicity assay. Environ Sci Technol 2013, 47:1110-1119. The study demonstrated the close correlations betweenin vitro data from fish cells and in vivo acute toxicity of fish, indicating the predictive power of the cell model RTgill-W1 to fish acute toxicity. This provides the basis of using cells in chemical risk evaluation of the aquatic environment. 12. Schirmer K: Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish. Toxicology 2006, 224:163-183. 13. Kramer NI, Hermens JLM, Schirmer K: The influence of modes of action and physicochemical properties of chemicals on the correlation between in vitro and acute fish toxicity data. Toxicol In Vitro 2009, 23:1372-1379. 14. Scholz S, Sela E, Blaha L, Braunbeck T, Galay-Burgos M, Garcı´a-Franco M, Guinea J, Klu¨ver N, Schirmer K, Tanneberger K et al.: A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment. Regul Toxicol Pharmacol 2013, 67:506-530. www.sciencedirect.com
Cell-culture-based biosensor for water toxicity Tan and Schirmer 67
15. Bury NR, Schnell S, Hogstrand C: Gill cell culture systems as models for aquatic environmental monitoring. J Exp Biol 2014, 217:639-650. 16. Dayeh VR, Schirmer K, Bols NC: Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent. Water Res 2002, 36:3727-3738. 17. Castan˜o A, Bols N, Braunbeck T, Dierickx P, Halder M, Isomaa B, Kawahara K, Lee LEJ, Mothersill C, Pa¨rt P et al.: The use of fish cells in ecotoxicology. The report and recommendations of ECVAM Workshop 47. Altern Lab Anim ATLA 2003, 31:317-351. 18. Segner H: Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. Altern Lab Anim ATLA 2004, 32:375-382. 19. Bols NC, Dayeh VR, Lee LEJ, Schirmer K: Chapter 2 use of fish cell lines in the toxicology and ecotoxicology of fish. Piscine cell lines in environmental toxicology [Internet]. Biochemistry and Molecular Biology of Fishes. Elsevier; 2005:: 43-84. 20. Dayeh VR, Bols NC, Tanneberger K, Schirmer K, Lee LEJ: The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD’s fish toxicity testing framework No. 171. In Curr Protoc Toxicol Editor. Board. Edited by Maines M. 2013. Chapter 1: Unit 1.5. 21. Giaever I, Keese CR: Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A 1984, 81:3761-3764. 22. Jiang WG (Ed): Electric Cell-substrate Impedance Sensing and Cancer Metastasis. Springer; 2012. 23. van der Schalie WH, James RR, Gargan TP: Selection of a battery of rapid toxicity sensors for drinking water evaluation. Biosens Bioelectron 2006, 22:18-27. 24. Widder MW, Trader D, Brennan L, Shedd TR, Gargan TP II, van der Schalie WH, Salazar N, Vallejo YR: An Environmental Sentinel Biomonitor System for Drinking Water Protection [Internet]. DTIC Document; 2008. 25. Curtis TM, Widder MW, Brennan LM, Schwager SJ, van der Schalie WH, Fey J, Salazar N: A portable cell-based impedance sensor for toxicity testing of drinking water. Lab Chip 2009, 9:2176-2183. 26. Brennan LM, Widder MW, Lee LEJ, van der Schalie WH: Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells. Toxicol Vitro Int J Publ Assoc BIBRA 2012, 26:736-745. 27. Widder MW, Brennan LM, Hanft EA, Schrock ME, James RR, van der Schalie WH: Evaluation and refinement of a field-portable drinking water toxicity sensor utilizing electric cell-substrate impedance sensing and a fluidic biochip: evaluation and refinement of a field-portable water toxicity sensor. J Appl Toxicol 2015, 35:701-708. This paper accounted some of the latest developments of the impedance-based biosensor developed by the US Army. The current prototype is a portable device using RTgill-W1 cells with optimised procedural steps, and reflects the essence of the decade-long pursuit of this line of biosensor. 28. Brennan LM, Widder MW, McAleer MK, Mayo MW, Greis AP, van der Schalie WH: Preparation and testing of impedance-based fluidic biochips with RTgill-W1 cells for rapid evaluation of drinking water samples for toxicity. J Vis Exp 2016 http://dx.doi. org/10.3791/53555. This work contains a step-by-step protocol of using the impedancebased biosensor developed by the US Army. The video and guide could be very useful for end users and developers of similar biosensors. 29. Pan T, Li H, Khare S, Huang B, Yu Huang D, Zhang W, Gabos S: High-throughput screening assay for the environmental water samples using cellular response profiles. Ecotoxicol Environ Saf 2015, 114:134-142. The study tested real water samples with xCELLigence using a variety of mammalian cell lines. It contains useful information of data evaluation and about environmental water sample monitoring on an impedance-based test platform. 30. Bohrn U, Mucha A, Werner CF, Trattner B, Ba¨cker M, Krumbe C, Schienle M, Stu¨tz E, Schmitt-Landsiedel D, Fleischer M et al.: www.sciencedirect.com
A critical comparison of cell-based sensor systems for the detection of Cr(VI) in aquatic environment. Sens Actuators B Chem 2013, 182:58-65. 31. Rakers S, Imse F, Gebert M: Real-time cell analysis: sensitivity of different vertebrate cell cultures to copper sulfate measured by xCELLigenceJ. Ecotoxicology 2014, 23:15821591. This study tested xCELLigence with mammalian and fish cell lines, and compared the data with conventional assays. It provides a good overview of how toxicity was manifested among different cell lines and reflected among different assays. 32. Kubisch R, Bohrn U, Fleischer M, Stu¨tz E: Cell-based sensor system using L6 cells for broad band continuous pollutant monitoring in aquatic environments. Sensors 2012, 12:3370-3393. 33. Liu F, Chen T, Zhang X, Li F, Voiculescu I: Study of Long Term Viability of Endothelial Cells on Biochip for Rapid and Reliable Water Toxicity Measurements. ASME; 2013:. V001T02A002V001T02A002. 34. Liu F, Li F, Nordin AN, Voiculescu I: A novel cell-based hybrid acoustic wave biosensor with impedimetric sensing capabilities. Sensors 2013, 13:3039-3055. 35. Liu F, Nordin AN, Li F, Voiculescu I: A lab-on-chip cell-based biosensor for label-free sensing of water toxicants. Lab Chip 2014, 14:1270-1280. This study showed that the combination of two biosensing mechanisms (impedance and quartz crystal microbalance resonator) resulted in increased accuracy of detection. It is a typical example of integrating multiparametric biosensors for enhanced performance. 36. Chaplen FW, Upson RH, Mcfadden PN, Kolodziej W: Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens. Pigment Cell Res 2002, 15:19-26. This is a pioneer study of chromatophore-based biosensors. Its development led to the successful application of a patent, a chain of events that were not often observed in biosensor development for water toxicity. Encouraging such initiatives and routes would help promote the adoptation and field application of biosensors for water monitoring. 37. Dierksen KP, Mojovic L, Caldwell BA, Preston RR, Upson R, Lawrence J, McFadden PN, Trempy JE: Responses of fish chromatophore-based cytosensor to a broad range of biological agents. J Appl Toxicol JAT 2004, 24:363-369. LV, Jovanovic GN: Development of a microbiosensor 38. Mojovic based on fish chromatophores immobilized on ferromagnetic gelatin beads. Food Technol Biotechnol 2005, 43:1-7. 39. Plant TK, Chaplen FW, Jovanovic G, Kolodziej W, Trempy JE, Willard C, Liburdy JA, Pence DV, Paul BK: Sensitive-cell-based Fish Chromatophore Biosensor. Edited by Mahadevan-Jansen A, Sowa MG, Puppels GJ, Gryczynski Z, Vo-Dinh T, Lakowicz JR.2004:265. 40. Sharma V, Narayanan A, Rengachari T, Temes GC, Chaplen F, Moon U-K: A low-cost, portable generic biotoxicity assay for environmental monitoring applications. Biosens Bioelectron 2005, 20:2218-2227. 41. Dukovcic SR, Hutchison JR, Trempy JE: Potential of the melanophore pigment response for detection of bacterial toxicity. Appl Environ Microbiol 2010, 76:8243-8246. 42. Dukovcic SR, Hutchison JR, Trempy JE: Conservation of the chromatophore pigment response. J Appl Toxicol 2010, 30:574581. 43. Iuga A, Lerner E, Shedd TR, van der Schalie WH: Rapid responses of a melanophore cell line to chemical contaminants in water. J Appl Toxicol 2009, 29:346-349. 44. Polak ME, Rawson DM, Haggett BGD: Redox mediated biosensors incorporating cultured fish cells for toxicity assessment. Biosens Bioelectron 1996, 11:1253-1257. 45. Wang S, Ramachandran A, Ja S-J: Integrated microring resonator biosensors for monitoring cell growth and detection of toxic chemicals in water. Biosens Bioelectron 2009, 24:3061-3066. Current Opinion in Biotechnology 2017, 45:59–68
68 Environmental biotechnology
46. Cevenini L, Calabretta MM, Tarantino G, Michelini E, Roda A: Smartphone-interfaced 3D printed toxicity biosensor integrating bioluminescent “sentinel cells”. Sens Actuators B Chem 2016, 225:249-257. 47. Bols NC, Barlian A, Chirino-Trejo M, Caldwell SJ, Goegan P, Lee LEJ: Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. J Fish Dis 1994, 17:601-611. 48. Dayeh VR, Grominsky S, DeWitte-Orr SJ, Sotornik D, Yeung CR, Lee LEJ, Lynn DH, Bols NC: Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena. Res Microbiol 2005, 156:93-103.
53. Schirmer K, Dixon D, Greenberg B, Bols N: Ability of 16 priority PAHs to be directly cytotoxic to a cell line from the rainbow trout gill. Toxicology 1998, 127:129-141. 54. Dayeh VR, Schirmer K, Bols NC: Ammonia-containing industrial effluents, lethal to rainbow trout, induce vacuolisation and Neutral Red uptake in the rainbow trout gill cell line, RTgill-W1. Altern Lab Anim ATLA 2009, 37:77-87. 55. Nano-Tera Swiss National Science Foundation: Envirobot. URL: https://wp.unil.ch/envirobot/. [Accessed 31 October 2016]. 56. Research consortium Envirobot: Envirobot. URL: http://www. nano-tera.ch/projects/345.php#desc. [Accessed 31 October 2016].
49. Lee LE, Dayeh VR, Schirmer K, Bols NC: Fish cell lines as rapid and inexpensive screening and supplemental tools for whole effluent testing. Integr Environ Assess Manag 2008, 4:372.
57. van der Meer JR: Towards improved biomonitoring tools for an intensified sustainable multi-use environment. Microb Biotechnol 2016, 9:658-665.
50. Glawdel T, Elbuken C, Lee LEJ, Ren CL: Microfluidic system with integrated electroosmotic pumps, concentration gradient generator and fish cell line (RTgill-W1)—towards water toxicity testing. Lab Chip 2009, 9:3243.
58. Chaplen F, Gerwick W, Jovanovic G, Kolodziej W, Liburdy JA, McFadden PN, Paul BK, Plant TK, Trempy JE, Willard C: Methods for detecting bioactive compounds. 2005, Patent No. US6913877 B1.
51. Lee LEJ, Dayeh VR, Schirmer K, Bols NC: Applications and potential uses of fish gill cell lines: examples with RTgill-W1. Vitro Cell Dev Biol—Anim 2009, 45:127-134.
59. Widder MW, Brennan LM, Trader D, Shedd TR, van der Schalie WH: Environmental Sentinel Biomonitor System (ESB). 2012, Patent Application No. US 13/156,826.
52. Yue Y, Behra R, Sigg L, Schirmer K: Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium. Nanotoxicology 2016, 10:1075-1083.
Current Opinion in Biotechnology 2017, 45:59–68
www.sciencedirect.com