WO2018212714A1

WO2018212714A1 - Toxicity testing device and methods for making and using the same

Info

Publication number: WO2018212714A1
Application number: PCT/SG2018/050237
Authority: WO
Inventors: Andrew Chwee Aun Wan; Tze Chiun Lim; Jia Kai LIM
Original assignee: Agency For Science, Technology And Research
Priority date: 2017-05-15
Filing date: 2018-05-15
Publication date: 2018-11-22

Abstract

A toxicity testing device and a method of making and using the device in toxicity testing is disclosed. The device including a three dimensional interfacial polyelectrolyte complexation (IPC) construct comprising vertebrate cells encased in a central compartment, wherein the cells are modified with a stressor gene (e.g. metallothionein) that is expressed in the presence of a toxin and a reporter gene downstream the stressor gene (e.g. SEAP, secreted form of embryonic alkaline phosphatase) comprising a green fluorescent protein (GFP). The device further comprises a first channel in fluid communication to a first side of the central compartment for providing culture media and a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

Description

Toxicity testing device and methods for making and using the same

Cross reference to related applications

[0001] This application claims the priority to Singapore application No. 10201703946Q, filed 15 May 2017, the contents of which are incorporated herein by reference.

Field

[0002] The present invention relates to a cell based toxicity testing device, preferably for testing air and water contamination and methods and kits for making and using such cell based toxicity testing devices.

Background

[0003] As air and water are vital resources, contamination of ambient air or water is an especially pressing issue. Ambient air or water contamination can occur due to the release of chemicals into the environment from homes or industry. While such release may be inadvertent or advertent, purposeful release of toxic chemicals or pathogens poses a particularly serious threat to the biosecurity of water sources and environments. Many adverse effects may arise from the exposure of humans to these air or water contaminants, at different levels [Nair R. et al. Toxicol. Appl. Pharmacol. 279 (2014), 338-350], Contaminated ambient air or water not only threatens the safety of those directly exposed during outdoor activities, in less developed regions, it may lead to a contaminated supply of drinking water. Where affected aquatic organisms form part of the food chain that ultimately leads back to humans, an indirect but equally serious health issue may emerge from water contamination. Despite the obvious need to keep a close watch on activities that could potentially contaminate ambient air or water, policing large areas of the atmosphere or water poses many challenges. For example, it is estimated that the water catchment area in Singapore alone makes up two-thirds of its land surface [https://www.pub.gov.sg/watersupply/fournationaltaps/localcatchmentwater] or an area of approximately 500 km2. Therefore, there is a pressing need for devices that enable the monitoring of ambient air and/or water quality.

[0004] While physical/chemical based sensors would be the ideal type of sensors to detect levels of a specific compound or physical property, the identity of the contaminant must be known before-hand. This would not always be the case, especially when the contamination is advertent. In the scenario where no information on the contaminant is available, the ideal sensor would be able to respond to any stimuli that compromises air and/or water quality. The type of sensor capable of meeting such a requirement is invariably one that is cell-based. Furthermore, the response of cell-based sensors may be related to the actual physiological response of exposed individuals.

[0005] Cell-based sensors can employ vertebrate, yeast or microbial cells [Poulsen A., et al. Urban Water Security Research Alliance Technical Report ISSN 1836-5566, March 201 1], Yeast and microbial cells are generally more robust than vertebrate cells, can be easily stored and reconstituted for use, and remain viable for long periods of time. They are thus ideal for applications in harsh environments, such as chemical waste effluents, or where the salinity or other environmental parameters preclude use of mammalian cells, such as ocean water. However, there are advantages in using vertebrate cells. Of note, vertebrate cells can respond to toxins, such as those of microbial origin, which microbial-based sensors are unable to detect [Banerjee and Bhunia Biosens. Bioelectron. 26 (2010), 99-106]. Vertebrate cell-based sensors can also provide readouts that are more physiologically relevant to animals and humans. If required, analysis of these cells following exposure to toxin can be performed, giving information regarding the mechanism of toxin action and its potency. Thus, vertebrate cell-based sensors make an excellent first line of screening or early warning system to counter threats to air and/or water quality.

[0006] A major challenge in developing vertebrate or human cell based-sensors is maintaining their viability for a prolonged period of time, under ambient conditions. This is deemed the ^"bottleneck" that has limited the on-site application of such sensors to date.

[0007] An object of the invention is to ameliorate some of the above mentioned difficulties.

Summary

[0008] A technology that provides a method to maintain the viability of vertebrate cells for a prolonged period of time under ambient conditions for toxicity testing by the cells is needed.

[0009] Accordingly, a first aspect of the invention includes a toxicity testing device comprising a) a 3 dimensional interfacial polyelectrolytes cornplexation (IPC) construct comprising vertebrate cells encased in a central compartment; b) a first channel in fluid communication with a first side of the central compartment for providing culture media; and c) a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested. [0010] Another aspect of the invention relates to a method of making a toxicity testing device comprising: a) contacting a polycation with a polyanion wherein vertebrate cells are contained in either the polycation or the polyanion prior to contact; b) drawing a fiber with an object at the interface of the polycation and the polyanion; c) forming a 3 dimensional interfacial polyelectrolyte complexation (IPC) construct from the fibers comprising vertebrate cells; d) placing the IPC construct into a central compartment; and e) adding components to the device that form a first channel in fluid communication with a first side of the central compartment for providing culture media and a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

[0011] Another aspect of the invention comprises a method of testing a fluid sample to determine if it is toxic comprising: a) passing culture media through a first channel in fluid communication with a first side of a central compartment containing a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells; b) passing the fluid sample through a second channel in fluid communication with a second side of the central compartment containing a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells for providing a sample to be tested; and c) detecting any changes in the vertebrate cells via a transparent portion of the central compartment; wherein a change in the vertebrate cells is indicative of the presence of a toxin in the fluid sample.

[0012] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

Brief Description of the Drawings

[0013] In the figures, which illustrate, by way of example only, embodiments of the present invention,

[0014] Figure 1 : A plan view of various examples of the toxicity testing device.

[0015] Figure 2: schematic of the method of making the toxicity testing device of Figure 1A. A An IPC construct (cell-laden IPC fibers) is formed and C inserted in a central compartment which is then placed in B the main unit whereby D the central compartment is flanked by two channels for perfusion of tissue culture media and water sample, respectively. D A peristaltic pump can be used to maintain constant flow conditions. The flow-through can be passed to the first inlet port and the second inlet port at the same time. [0016] Figure 3: schematic of the method of making the toxicity testing device of Figure

I B. A An IPC construct is formed and B inserted into the lumen of a tube. C T junctions are connected D to both ends of the tube for perfusion of tissue culture media and water sample, respectively. E Similar to the first design (Fig 1 A), a peristaltic pump can be used to maintain constant flow conditions.

[0017] Figure 4: schematic of the method of making the toxicity testing device of Figure

IC. A An IPC construct (cell-laden IPC fibers) is formed and B inserted in a central compartment flanked by two channels for perfusion of tissue culture media and water sample, respectively. C A transparent cover is then clamped on top and tightly apposed to the lower part to create a closed perfusion system. D Similar to the first design (Fig 1A), a peristaltic pump can be used to maintain constant flow conditions.

[0018] Figure 5: Cell viability under different conditions over 24 hours. Top panel: cells exhibit good viability in tissue culture media as shown by the LIVE/DEAD Viability/Cytotoxicity Kit. (green stain: live cells, red stain: dead cells) Middle panel: Majority of cells are not viable in water under static conditions. Bottom panel: cells exhibit good viability in device, when perfused simultaneously with water and tissue culture media.

[0019] Figure 6: A Perfused sample from the device under various conditions tested positive for alkaline phosphatase after different time periods. B Cells subject to perfusion conditions for 5 days exhibited excellent viability, as shown by the LIVE/DEAD Viability/Cytotoxicity Kit (green stain: live cells, red stain: dead cells).

[0020] Figure 7: HEK 293 cells transfected with plasmid encoding MT2A promoter driving SEAP expression respond to the presence of heavy metal ions (Hg, Pb, Cr). Y-axis indicates absorption of solutions containing the respective metal ions at 650 nm, following QUANTI-BlueTM assay for detection of SEAP activity.

[0021] Figure 8: Hybrid EF1 alpha-MT2A promoter: Letters in lower case denote part of the EFI alpha promoter (arising from EF1 alpha-exon1 ) and letters in upper case denote the human metallothionein 2A promoter. The AP-1 site (underlined) was mutated in the human metallothionein 2A promoter from GTGACTCAG to GTGTCTAGA (substituted bases in bold). This mutation greatly reduces basal transcriptional activity while allowing hyperinducibility upon treatment with heavy metal ions. The hybrid promoter consisting of a part of EFI alpha promoter and the modified metallothionein 2A promoter allows for a high transcriptional activation of downstream reporter gene. Detailed Description

[0022] Referring to Figure 1 , the device 10 of the current disclosure provides a method to maintain the viability of vertebrate cells for a prolonged period of time, under ambient conditions.

[0023] Accordingly, a first aspect of the invention includes a toxicity testing device comprising a) a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells encased in a central compartment; b) a first channel in fluid communication with a first side of the central compartment for providing culture media; and c) a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

[0024] The formation of a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells that is then centrally located between two channels in fluid connection with the IPC construct allows a user to simultaneously expose the sensing cells to tissue culture media and the test sample, while minimizing the degree to which they mix. Further, the incorporation of IPC fibers as a scaffold into the two-channel cell-based sensor overcomes the technical challenge of long-term maintenance of vertebrate cell viability in ambient condition, allowing for a higher density of cells while permitting efficient nourishment and sample testing.

[0025] One innovative aspect of the device is the encapsulation of the vertebrate cells which are sensing cells within the 3D matrix of fibers, by a process of interfacial polyelectrolyte complexation (IPC). Any method of interfacial polyelectrolyte complexation (IPC) known in the art that results in a seeding density of at least 10⁷ cells/ml would be suitable. Culture of cells using IPC fibers offers several advantages. Firstly, the ability to form microscale fibrous matrices under physiological conditions allows cells to be uniformly encapsulated in the fibers at high seeding density (~10⁷ ceils/ml) and excellent viability. The engineered assemblages of individual cell-fiber structures further exposes the encapsulated cells to soluble factors which facilitates efficient propagation of encapsulated cells. This cell culture platform can be readily scaled up, and is highly versatile in terms of the various compositions and conditions that can be used.

[0026] Three alternative designs of the device 10 are shown in Figure 1 wherein a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells 12 is encased in a central compartment 14. The central compartment 14 is in fluid communication with a first channel 16 on a first side 18 and is also in fluid communication with a second channel 20 on a second side 22 which is opposite the first side 18. The first channel 16 having a first inlet port 24 and a first outlet port 26 for a ceil culture media to be passed or flow though by entering from the first inlet port 24, passing the central compartment 14 that is in fluid communication with the first channel 16 allowing the culture media to enter central compartment 14 and pass into the 3 dimensional interracial polyelectrolytes complexation (IPC) construct comprising vertebrate cells 12 permitting exchange of nutrients and components to keep the cells alive and remove waste products of the cells. This exchanged media can then exit from the first outlet port 26. Similarly, the second channel 20 having a second inlet port 28 and a second outlet port 30 for a fluid sample to be passed or flow though by entering from the second inlet port 28, passing the central compartment 14 that is in fluid communication with the second channel 20 allowing the fluid sample to enter central compartment 14 and pass into the 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells 12 permitting the cells to come into contact with and react to the fluid sample. The fluid sample then exits from the second outlet port 30.

[0027] As used herein the term vertebrate cells may refer to cells taken or derived from any vertebrate animal. In various embodiments the vertebrate cells may be taken or derived from human, bovine, murine, porcine, zebrafish, crocodilian, gallus, vertebrates. In various embodiments the vertebrate cells may be stem cells. In various embodiments the vertebrate cells may be human mesenchymal stem cells. In various embodiments the vertebrate cells may be human embryonic stem cells. In various embodiments the vertebrate cells may be dermal fibroblasts. In various embodiments the vertebrate cells may be bovine pulmonary arterial endothelial cells. In various embodiments the vertebrate cells may be endothelial cells. In various embodiments the vertebrate cells may be epidermal keratinocytes. In various embodiments the vertebrate cells may be follicle dermal papilla cells. In various embodiments the vertebrate cells may be induced pluripotent stem cells. In various embodiments the vertebrate cells may be hepatocytes. In various embodiments the vertebrate cells may be human embryonic kidney cells. In various embodiments the vertebrate cells may be more than one type of cell.

[0028] In various embodiments at least a portion of the central compartment is formed of a transparent material to allow imaging of the vertebrate cells within the IPC construct.

[0029] The vertebrate cells include a gene construct of a reporter gene downstream of a stressor gene that is expressed in the presence of a toxin. This may be achieved by any of the genetic engineering methods known in the art. In various embodiments the gene construct may be introduced via vectors such as viral vectors, plasmids, nucleases such as TALENs and CRISPR assisted physically by electroporation, microinjection, viral infection such as using lentivirus, liposomes or by any other methods known in the art. Genetic modification of vertebrate cells or the use of lines that are osmotolerant may allow these cells to be used for sensing in saline environments.

[0030] In various embodiments genetically engineered sensing cells are incorporated into the device and exposure to toxins induces the expression of a detectable label, for example alkaline phosphatase secretion or fluorescence. Any suitable detection marker would suffice, however, optical detection markers are preferred. Some suitable optical detection markers include chemiluminescent detection assays, comprising firefly luciferase, new luciferases from other organisms such as the Gaussia luciferase, from Gaussia princeps. Luciola luciferases, either red or green emitting both from the firefly Luciola italica; and Cypridina luciferase from Vargula Hilgendorfi, beta lactamase, and green fluorescent protein. Another useful reporter system is secreted alkaline phosphatase (SEAP). SEAP differs from endogenous alkaline phosphatase in that it is heat stable and not inhibited by L- homoarginine. As a reporter, SEAP offers the advantage of monitoring gene activity without lysing the cells. SEAP catalyzes the hydrolysis of pNitrophenyl phosphate (pNpp), yielding a colorimetric readout at about 405 nm.

[0031] In various embodiments the reporter gene expresses a truncated form of human placental alkaline phosphatase preferably a secreted form of embryonic alkaline phosphatase (SEAP) sequence comprising a nucleotide sequence represented by SEQ ID

NO: 1.

[0032] The advantage of SEAP is that it's quantifiable and secreted being able to be detected in situ. SEAP is very simple and very convenient. In contrast using luciferase detection requires cell extract. As a reporter gene SEAP has numerous advantages, it is heat-stable, can be secreted into cell culture supernatant and can be monitored for kinetic studies without having to lyse the cells.

[0033] In various embodiments the reporter gene expresses a green fluorescence protein, preferably comprising a nucleotide sequence represented by SEQ ID NO: 2.

[0034] In a similar manner to SEAP green fluorescence protein can be detected in situ without requiring cell extraction. Unlike SEAP it is not secreted from the cell. The GFP signal, on the other hand, can be monitored directly within the cell by means of a fluorescent microscope or be measured using any fluorimeter. [0035] in various embodiments the stressor gene comprises a transcription factor binding site induced by the presence of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kp) when the NF-kp is activated, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3.

[0036] In this example the vertebrate cell itself expresses TNFa in response to bacterial products like iipopoiysaccharide or other bacteria products. This may be due to the nature of the cell or it may have been engineered to expresses TNFa in response to bacterial products.

[0037] In various embodiments the 3 dimensional interfacial polyelectrolytes compiexation (IPC) construct comprising vertebrate cells may include a combination of vertebrate cells comprising: sensor cells whereby the sensor cells include a gene construct of a reporter gene downstream of a stressor gene that is expressed in the presence of a toxin; and mesenchymal cells that express TNFa in the presence of bacterial products like Iipopoiysaccharide or other bacteria products.

[0038] In various embodiments the stressor gene comprises metallothionein.

[0039] Human cells have been reported to upregulate expression of specific genes upon exposure to environmental stressors, such as heavy metal ions and DNA damaging agents. Metalothionein is an examples of these stressor genes that detect heavy metal ions. By inserting the sequence of reporter proteins such as secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) downstream of the promoter of the metallothionein, in various empodiments, cells can be engineered to detect the presence of heavy metal ions in a fluid sample.

[0040] In various embodiments the gene construct further comprises Hybrid EF1 alpha- human metallothionein promoter nucleotide sequence represented by SEQ ID NO: 4.

[0041] In various embodiments the cells may be engineered to transiently express SEAP upon activation of the metallothionein promoter. For example the pY-SEAP may be modified by replacing part of the EFI alpha promoter (between Age I and Hindlll restriction sites) with the human metallothionein 2A (MT2A) promoter. This may be prepared via gene synthesis (Figure 8). In the example in Figure 8 the letters in lower case denote part of the EFI alpha promoter (arising from EF1 alpha-exon1 ) and letters in upper case denote the human metallothionein 2A promoter. The AP-1 site (underlined) was mutated in the human metallothionein 2A promoter from GTGACTCAG to GTGTCTAGA (substituted bases in bold). This mutation greatly reduces basal transcriptional activity while allowing hyperinducibility upon treatment with heavy metal ions. The hybrid promoter consisting of a part of EF1 alpha promoter and the modified metallothionein 2A promoter allows for a high transcriptional activation of downstream reporter gene.

[0042] In various embodiments the first channel 16 comprises a first inlet port 24 and a first outlet port 26 and is otherwise enclosed within the device; and the second channel 20 comprises a second inlet port 28 and a second outlet port 30 and is otherwise enclosed within the device.

[0043] The enclosure of the first and second channel has the advantage of excluding the chance of cross contamination from the air. In this way only the fluid sample is being examined for toxins.

[0044] In various embodiments the central compartment is detachable from the device.

[0045] This provides the advantage of being able to easily replace the 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate ceils if required.

[0046] Another aspect of the invention relates to a method of making a toxicity testing device comprising: a) contacting a polycation with a polyanion wherein vertebrate cells are contained in either the polycation or the polyanion prior to contact; b) drawing a fiber with an object at the interface of the polycation and the polyanion; c) forming a 3 dimensional interfacial polyelectrolyte complexation (IPC) construct from the fibers comprising vertebrate cells; d) placing the IPC construct into a central compartment; and e) adding components to the device that form a first channel in fluid communication with a first side of the central compartment for providing culture media and a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

[0047] In various embodiments the polycation is selected from any one of chitosan, water soluble chitosan, water soluble chitin, chitosan tetraethylorthosilicate, water soluble deacetylated chitin, methylated collagen, cationic conjugated polymers, and amphiphilic peptide. In various embodiments the polyanion is selected from any one of alginate, sodium alginate, Terpolymer, and chromatin. The resulting 3D matrix of fibers created by a process of interfacial polyelectrolyte complexation (IPC), allows for a much higher cell density within the same volume, which allows a measurable concentration of the analyte to be achievable.

[0048] In various embodiments the 3 dimensional interfacial polyelectrolytes complexation (IPC) from the fibers comprising vertebrate cells to form an IPC construct may be formed by any method known in the art including forming IPC fibers and/or assembling microscale cell-laden hydrogels such as IPC fiber assembly technique (IPC-FAST) and multi- interfacial polyelectrolyte complexation (MIPC) or both techniques employed separately or concurrently resulting in micro-patterned polyelectrolyte hydrogel-based construct. In various embodiments the fibers may be drawn through or immersed in consecutive solutions of calcium chloride and/or a physiological culture medium such as a buffer like PBS to enhance crosslinking between fibers resulting in a denser population of cells.

[0049J As a general principle in a first step two oppositely charged polyelectrolyte solutions are placed in contact, upon which a polyelectrolyte complex forms at the interface. This interfacial complex acts as a viscous barrier which prevents free mixing of the two solutions, allowing for IPC fiber formation. Subsequently, the interfacial complex is drawn or pulled by means of an object. This action disrupts the interface, leading to scattered domains of complexation that act as fiber nucleation sites.

[0050] In various embodiments the fiber may be draw with any suitable object at the interface of the polycation and the polyanion. Examples of suitable objects include such as a pair of forceps or pipette tips, however any long thin object that would be able to contact both the polycation and the polyanion solutions at the interface and then allow the polycation and the polyanion solutions to move towards one another and interact as the object is drawn or pulled away from the interface would suffice.

[0051] The advantage of a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells formed from a polycation with a polyanion is that the resulting gel matrix formed allows for decent diffusion rate of the culture media and the fluid test sample. The IPC construct allows a diffusion rate such that the cells all have access to the tissue culture media and the test sample while minimizing the degree to which they mix. A gel containing cells encased in collagen only, had such a low diffusion rate and did not permit either the sample or the culture media to enter any cells other than those at the periphery (data not shown). As such it seems necessary to have an interfacial polyelectrolytes complexation (IPC) construct with both a polycation and a polyanion to provide a suitable diffusion rate for the device to work well.

[0052] In comparison to 2D culture, a 3D culture of cells in IPC fibers allows a much higher density of cells within the same volume (-50 fold), which is critical to achieve a measurable concentration of the analyte. Furthermore, the permeable matrix of IPC fibers affords excellent mass exchange of nutrients and cell metabolites with the surrounding medium. The latter feature is especially important for the present application, where the cells must be simultaneously nourished by tissue culture medium and exposed to the fluid test sample in an efficient manner. This can be achieved by continuous perfusion of the fibers using a device of the current invention.

[0053] The device may be made in several ways. Here we describe three ways to make three devices. The first example depicted in Figure 2 was made of Polydimethylsiloxane (PDMS), by first designing a template and then 3D printing the template mold. Subsequently, PDMS was mixed with the curing agent and casted into the 3D printed template mold. Degassing was carried out in a vacuum chamber before overnight curing was carried out at 60°C. The PDMS devices were subsequently removed and sterilized by soaking in 70% ethanol before use. The device of this example has several separate components. The main components 34 in Figure 2B includes the first channel 20 and the second channel 16 and a central cavity 32. Having constructed the construct comprising vertebrate cells 12 as described herein and depicted in Figure 2A, the construct comprising vertebrate cells 12 is located into the central compartment 14, which in this example is cuboidal, see Figure 2C. The central compartment 14 with the construct comprising vertebrate cells 12 is then placed into the central cavity 32 of the main components 34. Adding these two components 14 and 34 to the device then form a first channel 16 in fluid communication with a first side 18 of the central compartment 14 for providing culture media and a second channel 20 in fluid communication with a second side 22 of the central compartment 14 for providing a sample to be tested. The first inlet port 24 is then placed at a first end 15 of the first channel 16; the first outlet port 26 is placed in the second end 17 of the first channel 16; The second inlet port is placed into the first end 19 of the second channel 20; and the second outlet port 30 is placed in the second end 21 of the second channel 20, See Figure 2D.

[0054] The second example depicted in Figure 3 was made in much the same way. By first designing a template and then 3D printing the template mold. Subsequently, PDMS was mixed with the curing agent and casted into the 3D printed template mold. Degassing was carried out in a vacuum chamber before overnight curing was carried out at 60°C. The PDMS devices were subsequently removed and sterilized by soaking in 70% ethanol before use. The device of this example also has several separate components. Having constructed the construct comprising vertebrate cells 12 as described herein, see Figure 3A, the construct comprising vertebrate cells 12 is located into the central compartment 14, which in this example is cylindrical, see Figure 3B. The first channel 16, in the form of T-junction tubing in this example, is fitted to the first side 18 of the central compartment 14 with the construct comprising vertebrate cells 12, see Figure 3C. Then a second channel 20, in the form of T- junction tubing in this example is fitted to the second side 22 of the central compartment 14 with the construct comprising vertebrate ceils 12. Referring to Figure 3D, adding these two T-junction tubing components to the central compartment then form a first channel 16 in fluid communication with a first side 18 of the central compartment 14 for providing culture media and a second channel 20 in fluid communication with a second side 22 of the central compartment 14 for providing a sample to be tested. The first inlet port 24 is formed from one side of the T junction tubing of the first channel 16; the first outlet port 26 is formed from the other side of the T junction tubing of the first channel 16; The second inlet port is formed from one side of the T junction tubing of the second channel 20; and the second outlet port 30 is formed from the other side of the T junction tubing of the second channel 20.

[0055] The third example depicted in Figure 4, was made as two separate parts as shown in Figure 4; the initial base component34 can be made of PDMS as described above, while the upper component 36 is preferably made of a suitable rigid, transparent polymer, such as poly(methylmethacrylate). Relevant parts of all 3 devices can be made of a transparent material in order to facilitate imaging of the IPC constructs and solutions in the channels by optical means, such as fluorescence microscopy. The device of this example also has several separate components. The base component 34 in Figure 4B includes the first channel 20 and the second channel 16 and a central cavity 14 which forms most of the central compartment 14. Additionally, at each corner a through-hole 31 is incorporated having a cavity on the underside to house a bolt. Having constructed the construct comprising vertebrate cells 12 as described herein and depicted in Figure 4A, the construct comprising vertebrate cells 12 is located into the central cavity 14 which forms most of the central compartment 14. Referring to Figure 4B and 4C, a bolt 33 is passed through each through-hole 31 from the underside of the base component 34 resulting in a bolt protruding from each comer of the base component 34. The first inlet port 24 is then placed at a first end 15 of the first channel 16; the first outlet port 26 is placed in the second end 17 of the first channel 16; The second inlet port is placed into the first end 19 of the second channel 20; and the second outlet port 30 is placed in the second end 21 of the second channel 20. A upper component 36 having corresponding through-holes in each corner is then placed over the bolts 33 and pushed down to be flush with the base component 34. The upper component forms one of the walls in the central compartment 14. Similarly, upper component 36 forms an upper wall of the first channel 16 and an upper wall of the second channel 20 minimizing any air contaminants from entering the system and affecting the vertebrate cells. Adding these two components 34 and 36 then form a first channel 16 in fluid communication with a first side 18 of the central compartment 14 for providing culture media and a second channel 20 in fluid communication with a second side 22 of the central compartment 14 for providing a sample to be tested. See Figure 4C. To hold all the components in place a nut 35 is tightened onto each bolt 33 to hold the device together.

[0056] In various embodiments at least a portion of the central compartment is formed of a transparent material. Examples of suitable transparent material may include Polydimethylsiloxane, poly(methylmethacrylate) or any other transparent material known in the art.

[0057] In various embodiments the polycation is a water soluble deacetylated chitin and the polyanion comprises sodium alginate.

[0058] The advantage of a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells formed from water soluble deacetylated chitin and sodium alginate is that the resulting gel matrix formed allows for an optimum diffusion rate of the culture media and the test sample. The IPC construct allows a diffusion rate such that the cells all have access to the tissue culture media and the test sample while minimizing the degree to which they mix.

[0059] In various embodiments the vertebrate cells include a gene construct of a reporter gene downstream of a stressor gene that is expressed in the presence of a toxin.

[0060] In various embodiments the vertebrate cells may be engineered in any manner known in the art or described herein.

[0061] In various embodiments the reporter gene expresses a truncated form of human placental alkaline phosphatase preferably a secreted form of embryonic alkaline phosphatase (SEAP) sequence comprising a nucleotide sequence represented by SEQ ID NO: 1 as described herein above.

[0062] In various embodiments the reporter gene expresses a green fluorescence protein, preferably comprising a nucleotide sequence represented by SEQ ID NO: 2 as described herein above.

[0063] In various embodiments the stressor gene comprises a transcription factor binding site induced in the presence of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-k(i) when NF-k|3 is activated, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3. [0064] NF-κΒ encompasses a family of transcription factors that play a pivotal role in the inflammatory response, cell proliferation, and survival. In the absence of stimulus such as tumor necrosis factor a (TNF-a), lipopolysaccharide, or interleukin 1 (IL1 ), F-κΒ is sequestered in the cytosol in its inactive form, complexed to the inhibitory proteins of the ΙκΒ family. An induction signal leads to the activation of the ΙκΒ kinase complex (IKK), which targets the inhibitor proteins for ubiquitylation and proteasome-mediated degradation. The subsequently unbound, NF-κΒ protein translocates into the cell nucleus, where it binds recognizable target DNA sequences, termed κΒ sites (GGGRNYYYCC, where R is purine, Y is pyrimidine, and N is any base SEQ ID NO: 3) and activates various genes, depending on the nature of the stimulus.

[0065] in various embodiments the stressor gene comprises metallothionein as described herein above.

[0066] In various embodiments the gene construct further comprises Hybrid EF1 alpha- human metallothionein promoter nucleotide sequence represented by SEQ ID NO: 4 as described herein above.

[0067] In various embodiments the vertebrate cells are derived from human embryonic kidney cells, as described herein above.

[0068] In various embodiments the device is formed from a rigid transparent polymer, preferably polymethylmethacrylate ) or polydimethylsiloxane.

[0069] Another aspect of the invention comprises a method of testing a fluid sample to determine if it is toxic comprising: a) passing culture media through a first channel in fluid communication with a first side of a central compartment containing a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells: b) passing the fluid sample through a second channel in fluid communication with a second side of the central compartment containing a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells for providing a sample to be tested; and c) detecting any changes in the vertebrate cells via a transparent portion of the central compartment; wherein a change in the vertebrate cells is indicative of the presence of a toxin in the fluid sample.

[0070] As used herein the term fluid refers to a liquid or a gas. The liquid may be water such as from water bodies, drinking water The gas may be for air quality to test for toxins in the air such as hazardous air pollutants known to cause cancer and other serious health impacts pollutants including volatile and semi-volatile organic compounds, heavy metals, carbon monoxide, lead, nitrogen dioxide, ozone, particles sulfur dioxide and others.

[0071] In various embodiments the fluid sample is a water sample.

[0072] In various embodiments the culture media may be any growth medium able to sustain ceil life. Examples may include fetal bovine serum (FBS); other defined serum replacements; Eagle's minimal essential medium (EMEM); Dulbecco modified Eagle's minimal essential medium; Minimum Essential Medium Eagle - alpha modification: or any other culture medium known in the art.

[0073] In various embodiments the culture media and the fluid sample are passed through the first and second channel simultaneously.

[0074] The continuous flow of the culture media and the fluid sample enables the living cells encapsulated in the IPC construct to be simultaneously nourished by the tissue culture medium and exposed to the test sample.

[0075] The vertebrate sensing cells are encapsulated within the 3D matrix of fibers, by a process of interfacial polyelectrolyte complexation (IPC) and placed in the central compartment of the device. Continuous flow of media and test sample in the two channels flanking the cell-laden fibers allows the cells to be simultaneously nourished by tissue culture medium and exposed to the test sample in an efficient manner. The vertebrate cells to be used can be engineered to secrete a compound that is amenable to detection, when exposed to toxins in air or water.

[0076] In various embodiments the vertebrate cells express a gene construct of a reporter gene downstream of a stressor gene in the presence of a toxin wherein detection of the expression of the reported gene is used to image the toxin.

[0077] Cells can be engineered to respond appropriately to a specific chemical insult and used in conjunction with the device of the present invention, which would allow their viability to be maintained under ambient conditions for a prolonged period of time.

[0078] In various embodiments the method of testing a fluid sample further comprising adding a pNitrophenyl phosphate dye to the culture media to detect excretion of an expressed reporter gene comprising a truncated form of human placental alkaline phosphatase into the IPC construct, imaging the expression of the reporter gene by a colour change, using a spectrophotometer, a camera or by eye, preferably the truncated form of human placental alkaline phosphatase is a secreted form of embryonic alkaline phosphatase (SEAP) comprising a nucleotide sequence represented by SEQ ID NO: 1.

[0079] In various embodiments the reporter gene comprises a green fluorescence protein imaged using a fluorimeter, preferably the reporter gene comprises a nucleotide sequence represented by SEQ ID NO: 2 as described herein above.

[0080] In various embodiments the stressor gene comprises a transcription factor binding site and is expressed when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kp) is activated in the presence of TNF|3 as described herein above, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3 as described herein above.

[0081] In various embodiments the fluid sample is a water sample and the stressor gene comprises metallothionein activated by the presence of a heavy metal in the water sample.

[0082] In various embodiments the fluid sample is an air sample and the stressor gene comprises metallothionein activated by the presence of a heavy metal in the air sample.

[0083] In various embodiments the gene construct further comprises Hydrid EF1 alpha- human metallothionein promotor nucleotide sequence represented by SEQ ID NO: 4 as described herein above.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0085] Throughout this document, unless otherwise indicated to the contrary, the terms "comprising', "consisting of", "having" and the like, are to be construed as non-exhaustive, or in other words, as meaning "including, but not limited to^".

[0086] Furthermore, throughout the specification, unless the context requires otherwise, the word "include^" or variations such as "includes" or "including" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0087] As used in the specification and the appended claims, the singular form "a", and "the" include plural references unless the context clearly dictates otherwise. Examples

[0088] Engineering of Cells for Sensor

[0089] Cells can be engineered to respond appropriately to a specific chemical insult and used in conjunction with the device of the present invention, which would allow their viability to be maintained under ambient conditions for a prolonged period of time. Human cells have been reported to upregulate expression of specific genes upon exposure to environmental stressors, such as heavy metal ions and DNA damaging agents. Examples of these stressor genes include the metallothionein and TNF-σ genes. By inserting the sequence of reporter proteins such as secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) downstream of the promoter of the stressor genes, cells can be engineered to detect the presence of the stressors. As a reporter gene, SEAP has numerous advantages, it is heat-stable, can be secreted into cell culture supernatant and can be monitored for kinetic studies without having to lyse the cells. The GFP signal, on the other hand, can be monitored directly by means of a fluorescent microscope.

[0090] Cells were transfected to transiently express SEAP upon activation of the metallothionein promoter. pY-SEAP (Addgene) was modified by replacing part of the EF1 alpha promoter (between Agel and Hind 111 restriction sites) with the human metallothionein 2A (MT2A) promoter that was obtained via gene synthesis (Appendix 1 ). The plasmid encoding MT2A promoter driving expression of SEAP was transfected into HEK 293 cells using Lipofectamine 2000 (Thermo Fisher).

[0091] 3 dimensional interfacial polyelectrolvtes complexation (IPC) construct comprising vertebrate cells

[0092] In the present disclosure, sodium alginate (Sigma-Aldrich) and water-soluble chitin (prepared by deacetylation of crab chitin from Sigma-Aldrich) were used as the polyanion and polycation, respectively.

[0093] In the first example HEK-Dual TNF-a cells (Invivogen) were encapsulated in IPC fiber constructs and used in the device.

[0094] In the second example the modified cells described above were encapsulated in IPC fiber constructs and used in the device.

[0095] Forming the devices [0096] Device 1

[0097] The first device was formed from Polydimethylsiloxane (PDMS) as depicted in Figure 2. First a template was designed using So!idworks software (Dassauit Systemes) and then 3D printed using Objet Connex 350 (Stratasys) to form a template mold. Subsequently, PDMS (SYLGARD® 184 silicone elastomer kit, Dow Corning) was mixed at 10:1 ratio with the curing agent and casted into the 3D printed template mold. Degassing was carried out in a vacuum chamber before overnight curing was carried out at 60°C. The PDMS devices were subsequently removed and sterilised by soaking in 70% ethanol before use.

[0098] Device 2

[0099] The second device was formed from Polydimethylsiloxane (PDMS) as depicted in Figure 3. First a template was designed using Solidworks software (Dassault Systemes) and then 3D printed using Objet Connex 350 (Stratasys) to form a template mold. Subsequently, PDMS (SYLGARD® 184 silicone elastomer kit, Dow Corning) was mixed at 10:1 ratio with the curing agent and casted into the 3D printed template mold. Degassing was carried out in a vacuum chamber before overnight curing was carried out at 60°C. The PDMS devices were subsequently removed and sterilised by soaking in 70% ethanol before use.

[00100] Device s

[00101] The third device was comprised of two separate parts as shown in Figure 4; the lower part can be made of PDMS as described above, while upper part is preferably made of a suitable rigid, transparent polymer, such as poly(methylmethacrylate).

[00102] Relevant parts of all 3 devices can be made of a transparent material in order to facilitate imaging of the IPC constructs and solutions in the channels by optical means, such as fluorescence microscopy.

[00103] Detection

[00104] TNF-cf detection in HEK-Dual J F-a cells

[00105] Continuous flow of media and test sample in the two channels flanking the cell-laden fibers can be maintained to preserve cell viability for a prolonged period of time under ambient conditions (Figure 5). To demonstrate the utility of the device in producing a measurable cell response upon exposure to a test sample, HEK- Dual TNF-σ cells (Invivogen) were encapsulated in IPC fiber constructs and used in the device. The latter cell line secretes alkaline phosphatase when exposed to toxins, via production of TNFo (produced by cells in response to toxins), which in turn activates the NF- k/? gene. The device was perfused with a fluid test samples containing varying concentrations of TNF-ff at a flow rate of 90 /L/min. After different time periods, samples of the perfusate were subject to QUANTI-BlueTM (Invivogen) assay to detect SEAP activity, resulting in different levels of a blue coloration (Figure 6A). Cells subject to these perfusion conditions for 5 days exhibited excellent viability (Figure 6B).

[00106] Heavy metal detection in engineered cells

[00107] To illustrate the utility of such an approach, cells were transfected to transiently express SEAP upon activation of the metallothionein promoter. pY- SEAP (Addgene) was modified by replacing part of the EF1 alpha promoter (between Age I and Hindil I restriction sites) with the human metallothionein 2A (MT2A) promoter that was obtained via gene synthesis (Figure 8). The plasmid encoding MT2A promoter driving expression of SEAP was transfected into HEK 293 cells using Lipofectamine 2000 (Thermo Fisher). After 1 day of transient transfection, heavy metal ions were added into the culture media at concentrations representing the maximum tolerable limit in either the sewage or watercourse in Singapore. [Retrieved from http://www.nea.gov.sg/anti-pollution-radiation- protection/water-pollution-control/allowable-limits] After 1 day of exposure to the heavy metal toxins, 20 /I of culture supernatant was added to 100/vl of QUANTI-BlueTM (Invivogen) to detect activity of SEAP that was induced by the heavy metal ions. Cells without exposure to heavy metal ions and cell culture media only were included as negative controls. The results showed that a measurable response could be obtained when the heavy metals were present, in contrast to the negative controls (Figure 7).

[00108] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.

Claims

Claims:

1. A toxicity testing device comprising:

a) a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells encased in a central compartment;

b) a first channel in fluid communication with a first side of the central compartment for providing culture media;

c) a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

2. The device according to claim 1 , wherein at least a portion of the central compartment is formed of a transparent material to allow imaging of the vertebrate cells within the IPC construct.

3. The device according to claim 1 or 2, wherein the vertebrate cells include a gene construct of a reporter gene downstream of a stressor gene that is expressed in the presence of a toxin.

4. The device according to claim 3, wherein the reporter gene expresses a truncated form of human placental alkaline phosphatase preferably a secreted form of embryonic alkaline phosphatase (SEAP) comprising a nucleotide sequence represented by SEQ ID NO;

1.

5. The device according to claim 3, wherein the reporter gene expresses a green fluorescence protein, preferably comprising a nucleotide sequence represented by SEQ ID

NO: 2.

6. The device according to any one of claims 3 to 5, wherein the stressor gene comprises a transcription factor binding site induced by the presence of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-kp) when the NF-kp is activated, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3.

7. The device according to any one of claims 3 to 5, wherein the stressor gene comprises metallothionein.

8. The device according to any one of claims 3 to 6, wherein the gene construct further comprises Hybrid EF1 alpha-human metallothionein pro motor nucleotide sequence represented by SEQ ID NO: 4.

9. The device according to any one of claims 1 to 8, wherein the first channel comprises a first inlet port and a first outlet port and is otherwise enclosed within the device; and the second channel comprises a second inlet port and a second outlet port and is otherwise enclosed within the device.

10. The device according to any one of claims 1 to 9, wherein the central compartment is detachable from the device.

1 1 . A method of making a toxicity testing device comprising:

a) contacting a polycation with a polyanion wherein vertebrate cells are contained in either the polycation or the polyanion prior to contact;

b) drawing a fiber with an object at the interface of the polycation and the polyanion; c) forming a 3 dimensional interfacial polyelectrolyte complexation (IPC) construct from the fibers comprising vertebrate cells:

d) placing the IPC construct into a central compartment; and

e) adding components to the device that form a first channel in fluid communication with a first side of the central compartment for providing culture media and a second channel in fluid communication with a second side of the central compartment for providing a sample to be tested.

12. The method according to claim 1 1 , wherein at least a portion of the central compartment is formed of a transparent material.

13. The method according to claim 1 1 , wherein the polycation is a water soluble deacetylated chitin and the polyanion comprises sodium alginate.

14. The method according to claim 1 1 , wherein the vertebrate cells include a gene construct of a reporter gene downstream of a stressor gene that is expressed in the presence of a toxin.

15. The method according to claim 14, wherein the reporter gene expresses a truncated form of human placental alkaline phosphatase preferably a secreted form of embryonic alkaline phosphatase (SEAP) sequence comprises a nucleotide sequence represented by SEQ ID NO: 1.

16. The method according to claim 14, wherein the reporter gene expresses a green fluorescence protein, preferably comprising a nucleotide sequence represented by SEQ ID

NO: 2.

17. The method according to any one of claims 14 to 16, wherein the stressor gene comprises a transcription factor binding site induced in the presence of nuclear factor kappa- light-chain-enhancer of activated B cells (NF-k[5) when NF-kp is activated, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3.

18. The method according to any one of claims 14 to 16, wherein the stressor gene comprises metallothionein.

19. The method according to any one of claims 14 to 17, wherein the gene construct further comprises Hybrid EF1 alpha-human metallothionein promotor nucleotide sequence represented by SEQ ID NO: 4.

20. The method according to any one of claims 1 1 to 19, wherein the vertebrate cells are derived from human embryonic kidney cells.

21 . The method according to any one of claims 1 1 to 20, wherein the device is formed from a rigid transparent polymer, preferably poly(methylmethacrylate), and a flexible polymer, preferably polydimethylsiloxane (PDMS).

22 A method of testing a fluid sample to determine if it is toxic comprising:

a) passing culture media through a first channel in fluid communication with a first side of a central compartment containing a 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate ceils;

b) passing the fluid sample through a second channel in fluid communication with a second side of the central compartment containing the 3 dimensional interfacial polyelectrolytes complexation (IPC) construct comprising vertebrate cells for providing a sample to be tested: and c) detecting any changes in the vertebrate cells via a transparent portion of the central compartment, wherein a change in the vertebrate cells is indicative of the presence of a toxin in the fluid sample.

23. The method according to claim 22, wherein the fluid sample is a water sample.

24. The method according to claim 22 or 23, wherein the culture media and the fluid sample are passed through the first and second channel simultaneously.

25. The method according to any one of claims 22 to 24, wherein the vertebrate cells express a gene construct of a reporter gene downstream of a stressor gene in the presence of a toxin wherein detection of the expression of the reported gene is used to image the toxin.

26. The method according to claim 25, further comprising adding a pNitrophenyl phosphate dye to the culture media to detect excretion of an expressed reporter gene comprising a truncated form of human placental alkaline phosphatase into the IPC construct, imaging the expression of the reporter gene by a colour change, using a spectrophotometer, a camera or by eye, preferably the truncated form of human placental alkaline phosphatase is a secreted form of embryonic alkaline phosphatase (SEAP) comprising a nucleotide sequence represented by SEQ ID NO: 1.

27. The method according to claim 25, wherein the reporter gene comprises a green fluorescence protein imaged using a fluorimeter, preferably the reporter gene comprises a nucleotide sequence represented by SEQ ID NO: 2.

28. The method according to any one of claims 25 to 27, wherein the stressor gene comprises a transcription factor binding site that is induced by nuclear factor kappa-light- chain-enhancer of activated B cells (NF-k(j) when the NF- kp is activated in the presence of TNFcc, preferably the transcription factor binding site comprises a nucleotide sequence represented by SEQ ID NO: 3.

29. The method according to any one of claims 25 to 28, wherein the stressor gene comprises metallothionein activated by the presence of a heavy metal in the fluid sample.

30. The method according to any one of claims 25 to 28, wherein the gene construct further comprises Hybrid EF1 alpha-human metallothionein pro motor nucleotide sequence represented by SEQ ID NO: 4.