Electrical measurements in a multilayered cell culture device

Abstract

A cell culture device comprising a multilayer stack and a sensor (30) disposed between the layers (12) of the multilayer stack (10).

Description

ELECTRICAL MEASUREMENTS IN A MULTILAYERED CELL CULTUREDEVICEFIELD OF INVENTION

[0001] The present invention relates to a cell culture device comprising sensors for electrical measurements.BACKGROUND OF INVENTION

[0002] Gut microbiome and gut-derived metabolites play key roles in biotransformation of drugs, by directly or indirectly influencing drug absorption, toxicity, and bioavailability in relationship with other cells or organs. In this domain, the gut barrier integrity needs to be evaluated, for instance using electrical measurements. Ex vivo cell co-cultures are a powerful tool to evaluate the reaction of a human or animal organ to an active principle such as a xenobiotic, a cosmetic, a drug. Microfluidic devices are extensively used to reproduce an environment favorable to the development and organization of cells, close to that of a tissue or an animal or a human organ.

[0003] However, known devices and methods for electrically evaluating the gut barrier integrity during cell culture are not satisfactory.

[0004] Indeed, known devices perform cell co-culture in superposed chambers separated by semi-permeable layers. Electrodes are placed on glass or polycarbonate substrates disposed above or below the chambers. Therefore, the electrical measurement signal comprises contribution from the whole device, including several cell cultures. It is impossible to use the co-culture device when placing the glass or polycarbonate substrate comprising electrodes between the chambers because of leakage and breaking of the substrate or electrodes. Moreover, when the substrate is placed between the chambers, it is superposed to the semi-permeable membrane. The substrate thus closes the permeability between the chambers.

[0005] Other devices use electrodes directly deposited onto the organ-on-a-chip layers. However, this imposes the manufacturing of electrodes with (at the same time) the device thereby limiting the manufacturing factors (temperatures, chemical) and exposing the organ-on-a-chip components to toxic chemicals requiring extensive cleaning procedures.

[0006] There is thus a need for a co-culture microfluidic device allowing gut barrier integrity evaluation without any leakage, which is easy to manufacture and which allows modularity when assembling the device.

[0007] To this end, a purpose of this invention is to provide a cell culture device comprising a multilayer stack comprising at least three gasket layers - each comprising at least one channel - and at least two semi-permeable layers, the gasket layers and the semi- permeable layers being alternating. Neighboring gasket layers are superposed so that at least part of the channels are aligned. The cell culture device further comprises an upper support and a lower support, the multilayer stack being disposed between the upper support and the lower support, and a sensor tight sealed to the multilayer stack and comprising at least four electrodes on a carrier film and connected to a connecting lead. Each carrier film is disposed on one of the layers of the multilayer stack and the electrodes are aligned with the channel of at least one of the gasket layers.SUMMARY

[0008] This invention thus relates to a cell culture device comprising: a multilayer stack comprising at least five layers wherein: o at least three of the layers are gasket layers, each gasket layer comprising at least one channel, each channel comprising an inlet and an outlet; and o at least two of the layers are semi-permeable layers; wherein the gasket layers and the semi-permeable layers are alternating; and wherein neighboring gasket layers are superposed so that at least part of the channels are aligned;an upper support and a lower support, the multilayer stack being disposed between the upper support and the lower support; and a sensor comprising at least four sensing units, each sensing unit being on a carrier film, each sensing unit comprising an electrode connected to a connecting lead, each carrier film being disposed on one of the layers of the multilayer stack, the electrodes being aligned with the channel of at least one of the gasket layers, wherein the sensor and the multilayer stack are tight sealed.

[0009] Indeed, the device of the invention allows an electrical measurement through only one type of cell thanks to the possibility to insert the sensor between the channels (chambers) while maintaining the sealing.

[0010] The upper and lower supports allow the application of a uniform pressure in order to tight seal the multilayer stack. Moreover, they allow the alignment of the layers. Finally, the upper and lower supports allow to close the channels from the top and bottom of the device.

[0011] According to an advantageous aspect of the invention, the gasket layer is elastomeric.

[0012] The elastomeric material provides the elasticity needed to provide a deformation of the gasket around the sensor therefore improving the tight sealing.

[0013] According to an advantageous aspect of the invention, the carrier film is a polyimide film, preferably an adhesive polyimide film.

[0014] The polyimide allows to provide thermal stability and high mechanical properties.

[0015] According to an advantageous aspect of the invention, the carrier film has a thickness ranging from 1 pm to 100 pm.

[0016] This thickness is small enough to increase the deformation of the gasket layers therefore improving the tight sealing.

[0017] According to an advantageous aspect of the invention, the sensing units have a thickness ranging from 1 nm to 500 nm.

[0018] Contrarily to commonly used electrodes which are very thick leading to leakage, the electrodes of the invention and their connecting leads have a thickness small enough to allow the deformation of the gasket layers around the sensing units therefore improving the tight sealing.

[0019] According to an advantageous aspect of the invention, the sensing units form a pair of sensing units, the electrodes of a pair of sensing units being opposite on either side of one of the channels.

[0020] According to an advantageous aspect of the invention, the sensing units are deposited on the carrier film.

[0021] The deposition allows to reach a thickness small enough to allow the deformation of the gasket layers around the sensing units therefore improving the tight sealing.

[0022] According to an advantageous aspect of the invention, the cell culture device further comprises at least one fastener configured to apply pressure on the multilayer stack.

[0023] The use of a fastener allows a better control of the pressure and a better distribution of the pressure on the multilayer stack.

[0024] According to an advantageous aspect of the invention, each channel comprises an inlet part comprising the inlet, an outlet part comprising the outlet and a linear part between the inlet part and the outlet part.

[0025] The linear part allows to avoid interferences between the electrical measurements.

[0026] According to an advantageous aspect of the invention, each channel comprises a circular part between the inlet and the outlet.

[0027] According to an advantageous aspect of the invention, the cell culture device further comprises a base, the lower support lying on the base.

[0028] The base provides additional support and a better alignment of the layers of the multilayer stack and the upper and lower supports. Moreover, the base may comprise spring loaded pogo pins configured to apply a pressure on the extremity of the connecting lead extending outside the multilayer stack in order to increase the efficiency of the electrical connection with the measuring unit.

[0029] The invention also relates to a cell culture system comprising: the cell culture device as described previously; a pump fluidly connected to the inlet and outlet of each channel for providing a cell culture medium to the channels; a measuring unit configured to receive signals from the electrodes.

[0030] According to an advantageous aspect of the invention, the cell culture system further comprises a bubble trap.

[0031] The bubble traps allow to reduce interferences in the measurements.

[0032] The invention also relates to a method of monitoring a cell culture, the method comprising the steps of: providing any of the cell culture device as described previously; establishing a cell culture in at least one of the channels, the electrodes being aligned with said channel; performing an electrical measurement with the sensor.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows an exploded view of a cell culture device according to an embodiment.

[0034] Figure 2 shows the basic embodiment (3 chambers) and the improved embodiment (4 chambers) in use.

[0035] Figure 3 shows the assembly steps of a cell culture device according to an embodiment.

[0036] Figure 4 shows gasket layers with electrodes and carrier film.

[0037] Figure 5 shows the manufacturing steps of the sensor.

[0038] Figure 6 shows the impedance measured for 12 days of culture.

[0039] Figure 7 shows the impedance measured before and after the cell barrier disruption.

[0040] Figure 8 shows the impedance measured before and after adding Calcium for cell barrier reconstruction.DEFINITIONS

[0041] In the present invention, the following terms have the following meanings:

[0042] “Cell culture parameter” refers to a parameter that affects cell culture growth and well-being. Accordingly, the parameter may relate to one or more parameters of the cell culture media, such as oxygen level, carbon dioxide level, pH, temperature, fluid flow rate, and / or the concentration of a substance, such as a nutrient or salt, suspended in the cell culture media. Cell culture parameter may also refer to a parameter of the growing cells, and may include cell number, cell density, electrical parameter such as electrical resistance. Accordingly, depending on the application of interest, such as cell type, one or more cell culture parameters may be monitored, including for use in an automated system wherein action may be taken so as to maintain a desired cell culture parameter for optimum growth, continued growth, and cell maintenance. Furthermore, any of the cell culture parameters may be used to signal when desired cell state, such as cell growth, has been achieved.

[0043] “Chamber” refers to the portion of the cell culture device where cells are placed and cultured. There may be an exchange or transfer of one or more chemicals or biological materials between chambers, including via diffusion or mass transport through the semi- permeable layers, but generally cells are confined in their own chamber.

[0044] “Deformable” refers to layers of the device, in particular gasket layers, that are able to deform under pressure in the direction normal to their surface (bending) in order to comply around an uneven element, such as electrodes, or uneven adjacent layer.

[0045] “Sealed” or “tight sealed” refers to the propriety of at least two materials which have a close contact of avoiding any leakage of aqueous liquid between them. In the Z- shape configuration, the sealed property is reached when any leakage is avoided under a pressure of 5.2 Bars during a minimum of 7 days.DETAILED DESCRIPTION

[0046] This invention relates to a cell culture device which is for example represented in figure 1. The cell culture device comprises: a multilayer stack 10; an upper support 21 and a lower support 23, the multilayer stack 10 being disposed between the upper support 21 and the lower support 23; and a sensor 30 comprising at least four sensing units.

[0047] A cell culture is a process in which cells 70 are grown ex vivo under controlled conditions. This allows to reproduce a cell structure close to that of a tissue or an animal or human organ in order to perform experiments, analysis and evaluation outside the human or animal body.

[0048] In particular, this allows to study the interactions between human or animal cells, for example gut epithelial cells, and bioactive molecules released by another type of cells, for example bacteria. In this example, this allows to study the interaction between the microbiota and the gut. The intestinal epithelial cells derived from cancerous cell lines orpatient-derived gut organoids are preferably cultured as a monolayer, i.e., with one layer of cells (no vertical superposition).

[0049] To do so, the multilayer stack 10 comprises at least five layers wherein: at least three of the layers are gasket layers 12; and at least two of the layers are semi-permeable layers 11.

[0050] The layers (11, 12) have a length, a width and a thickness, the thickness being small compared to the length and the width. The layers (11, 12) may have a polygonal section or a circular section. The top and the bottom of the layers (11, 12) are the surfaces of the layer on either side of the thickness.

[0051] The thickness of the gasket layers 12 ranges preferably from 0.2 mm to 2 mm. As better represented in figures 4A and 4B, each gasket layer 12 comprises at least one channel 13. The channel 13 may correspond to a furrow passing through the overall thickness of the gasket layer 12. In other words, the channel 13 is opened in the top and the bottom of the gasket layer 12. The height of the channel thus corresponds to the thickness of the gasket layer 12. The volume of a channel 13 may range from 10 pL to 5000 pL, preferably ranging from 210 pL to 870 pL. The surface of the channel 13, i.e., the projection of the channel 13 on the top or the bottom of the gasket layer 12 may range from 100 mm2to 1200 mm2. Each channel 13 comprises an inlet 13a and an outlet 13b allowing to dispose the cells in the channel 13 or to provide a fluid such as nutrient. Preferably, each channel 13 comprises a different type of cell to be cultured. The inlet 13a and outlet 13b may be the extremities of the channel 13 or may be in the form of holes in the gasket layer 12 separated from the channel 13 as in figure 4B. In this embodiment (figure 4B), an inlet duct 13c and an outlet duct 13c allow the fluidic communication between the inlet and outlet holes (13a, 13b) and the channel 13.

[0052] The gasket layer 12 is made of material allowing deformation / flexibility under pressure in the direction of the thickness. In other words, the gasket layer 12 is deformable. For example, the gasket layer 12 is elastomeric. Preferably, the gasket layer 12 is a rubber such as a silicone rubber.

[0053] The semi-permeable layer 11 is a layer permeable to molecules but not to cells. The thickness of the semi-permeable layer 11 ranges from lOnm to 10pm. For example, the semi-permeable layer 11 is a microporous membrane. The size of the pores ranges from 0.01 pm to 5 pm, preferably from 0.05 pm to 1 pm.

[0054] The gasket layers 12 and the semi-permeable layers 11 are alternating thereby forming a sandwich comprising successively a gasket layer 12, a semi-permeable layer 11, a gasket layer 12, a semi-permeable layer 11 and a gasket layer 12. For reasons of clarity, the semi-permeable layers 11 are not represented in figure 1 but are present between the gasket layers 12. In the following, the stacking direction is named the vertical direction. The number of layers may be increased by adding a gasket layer 12 separated from another gasket layer 12 by an additional semi-permeable layer 11. In the following, the gasket layer 12 at the bottom of the multilayer stack 10 when the device is in use is named the first gasket layer whereas the gasket layer 12 at the top of the multilayer stack 10 when the device is in use is named the last gasket layer.

[0055] Each semi-permeable layer 11 thus closes the top or the bottom of the channel 13 on which it is placed.

[0056] In a basic embodiment wherein the multilayer stack 10 comprises five layers (11, 12), the cell culture device comprises three channels, i.e., three cell culture chambers as represented in figures 2A. The lower channel 15a, in the first gasket layer 12, may be a perfusion channel wherein the culture medium is flowing. The intermediate channel 15b, superposed to the lower channel 15a, may be a cell culture chamber comprising, for example, the gut epithelial cells or gut organoids 70. The cells 70 of the cell culture chamber 15b are provided with the culture medium of the perfusion channel 15a through the semi-permeable layer 11. The upper channel 15d, in the last gasket layer 12, may comprise bioactive chemicals and / or different type of cells 70 releasing bioactive materials interacting with the cells 70 of the cell culture chamber 15b through the semi- permeable layer 11. The upper channel 15d comprises for example a microbiome, prebiotics or bacteria.

[0057] The following example lists the type of cells 70 that may be co-cultured in the basic embodiment (cells in the intermediate channel 15b / upper channel 15d): probiotics in Evaa and Antiallergic Air Optimizer (PBGL) / Calu-3 cells and nasal epithelial cells. Moreover, in the basic embodiment, the intermediate channel 15b may comprise cultured cells 70 such as Caco-2 cells (cancerous cell line) or gut organoids, the lower and upper channels (15a, 15d) not comprising cells.

[0058] In an improved embodiment wherein the multilayer stack 10 comprises seven layers, the cell culture device comprises four channels, i.e., four cell culture chambers as represented in figures 1 and 2B. The lower channel 15a, in the first gasket layer 12, may comprise a first cell type, for example immune, endothelial or neural cells 70. The first intermediate channel 15b, superposed to the lower channel 15a, may be a cell culture chamber comprising, for example, the gut epithelial cells or gut organoids 70. The second intermediate channel 15c, above the first intermediate channel 15b, may comprise bioactive chemicals and / or different type of cells 70 releasing bioactive materials for example a microbiome, prebiotics or bacteria, releasing bioactive chemicals interacting with the cells 70 of the cell culture chamber 15b through the semi-permeable layer 11. The cells 70 of the second intermediate layer 15c may be anaerobic organisms (anaerobes). The upper channel 15d, in the last gasket layer 12, may be filed with inert gas such as nitrogen in order to provide an anaerobic environment. Therefore, in the different layers, strict anaerobic (such as Clostridia), facultative anaerobic (such as Limosilactobacillus reuteri and Escherichia coli) and aerobic microbiota or mixture thereof in the system can be co-cultured.

[0059] The following examples list the type of cells 70 that may be co-cultured in the improved embodiment (cells in the lower channel 15a / first intermediate channel 15b / second intermediate channel 15c): peripheral blood mononuclear cell (PBMC) / Caco-2 cells (cancerous cell line) / Clostridia;Enteric nervous system cells (ENS) / Caco-2 cells (cancerous cell line) / Limosilactobacillus reuteri\ orHuman umbilical vein endothelial cells (HUVECs) and THP-1 cells / Caco-2 cells (cancerous cell line) / Escherichia coli.

[0060] Optionally, if the height of the channel 13 has to be increased, the gasket layers 12 may be formed by two superposed sublayers without semi-permeable layer 11 between them.

[0061] A coating 14, for example a collagen, mucin or matrigel coating, may be applied on the surface of a semi-permeable layer 11 placed below a gasket layer 12. The coating advantageously provides an optimal bed on which the cells 70 are cultured. It also allows to increase the adhesion of the cells to the semi-permeable layer 11.

[0062] Neighboring gasket layers 12 (separated by a semi-permeable layer 11) are superposed so that at least part of the channels 13 are aligned. Indeed, this allows the bioactive molecules produced by one type of cells 70 in a channel 13 to be transferred to the neighboring channel 13 through the semi-permeable layer 11.

[0063] In order to reach the inlet 13a and the outlet 13b of the channel 13 comprised in the gasket layer 12 between the first and the last gasket layers 12, each layer (11, 12) placed between said gasket layer 12 and the first or the last gasket layer 12 may comprise a communication hole 12a as represented in figure 1 allowing a fluidic communication to said inlet 13a and outlet 13b from the first or the last gasket layer 12. Preferably, the communication holes 12b are positioned in order to be aligned on the inlet 13a or the outlet 13b when the layers (11, 12) are assembled therefore forming an inlet pit and an outlet pit.

[0064] The upper and lower support (21, 23) provide rigidity to the whole multilayer stack 10. The upper support 21 may be placed on the top of the multilayer stack 10, z.e., above the last gasket layer 12, when the device is in use, the lower support 23 being thus placed on the bottom of the multilayer stack 10, i.e., below the first gasket layer 12. Therefore, the lower support 23 allows to close the bottom of the channel 13 of the fist gasket layer 12 (at the bottom of the multilayer stack 10) and the upper support 21 allows to close the top of the channel 13 of the last gasket layer 12 (at the top of the multilayer stack 10). The upper layer 21 may comprise at least one inlet port and / or at least oneoutlet port 21a to provide a fluidic entry and exit to the multilayer stack 10. The inlet and outlet ports 21a are thus preferably in fluidic communication with the inlet 13a and the outlet 13b of each channel 13. The upper and lower support (21, 23) may be polycarbonate lids. The upper and lower support (21, 23) may have a thickness ranging from 1mm to 10mm.

[0065] The lower support 23 may comprise alignment pins 23a, preferably six alignment pins 23a, extending in the vertical direction. In this embodiment, each of the layers (11, 12) of the multilayer stack 10 and the upper support 21 comprises openings 80 positioned in order to cooperate with the alignment pins 23a. When assembling the device, each layer (11, 12) is laid on the lower support 23, the alignment pins 23a engaging the openings 80 as shown in figure 3. This allows a better alignment between the layers (11, 12). Of course, alternatively, the alignment pins 23a may be on the upper support 21, the lower support 23 comprising openings 80.

[0066] The analysis and evaluation of the cells 70 cultured in the channels 13 is performed thanks to the sensor 30 better represented in figure 4. Each of the sensing unit of the sensor 30 comprises an electrode 34 connected to a connecting lead 36 allowing to transfer the signal measured by the electrodes 34 to a measuring unit. The sensing units are placed on a carrier film 32. The electrodes 34 and their connecting leads 36 may comprise Platinum, Titanium or Gold. Preferably, the electrodes 34 and their connecting leads 36 may comprise Platinum. Indeed, Platinum is less prone to fouling than Gold. Fouling refers to the accumulation of unwanted materials on the surface of a material or object, which can lead to reduced performance or functionality. A layer of Tantalum or Titanium may be placed between the carrier film 32 and the Platinum electrodes 34 and connecting traces 36. Indeed, Tantalum and Titanium allow to increase the adhesion of the Platinum on the carrier film 32. Moreover, contrarily to Chromium, Tantalum and Titanium do not have negative effects on the cell culture.

[0067] The carrier film 32 comprising the sensing units is placed in the multilayer stack 10, i.e., on one of the layers (11, 12) of the multilayer stack 10. The carrier film 32 may be placed between the lower support 23 and the first gasket layer 12, between two gasket layers 12, between a gasket layer 12 and a semi-permeable layer 11 or between thelast gasket layer 12 and the upper support 21. In order to measure a signal from the cells 70 cultured in the channel 13, the electrodes 34 are aligned with the channel 13 of at least one of the gasket layers 12. When the electrodes 34 are in direct contact with the cells, a coating is preferably applied on the electrodes 34. Preferably, the extremity of the connecting lead 36 opposite to the electrode 34 extends outside the multilayer stack 10 as in figure 1. This allows to easily connect the electrodes 34 to a measuring unit. The connection between the connecting lead 36 and the measuring unit may be ensured by a connecting pad 37.

[0068] The electrodes 34 are able to measure a signal on a linear channel (figure 4B) or on a circular channel (figure 4A). Preferably, the measure is performed on a linear part of the channel 13 because this reduces the interferences between the electrodes 34. For example, as represented in figure 4B, the channel 13 has a Z- shape comprising a linear part, therefore comprising two extremities, an inlet part connecting one extremity of the linear part to the inlet 13a and an outlet part connecting the other extremity of the linear part to the outlet 13b. For example, the volume of the Z- shape channel 13 ranges from 190 pL to 300 pL, preferably from 210 pL to 270 pL. The volume of the circular channel 13 may range from 500 pL to 1000 pL, preferably from 630 pL to 870 pL.

[0069] The sensing units preferably form a pair of sensing units. Each pair of sensing units may comprise an exciting electrode 34E and a measuring electrode 34M. The electrodes 34 of a pair of sensing units are opposite on either side of one of the channels 13 as represented in figure 2A. This allows impedance measurements. Preferably, two pairs of sensing units are placed so that the exciting electrodes 34E of the two pairs of sensing units are aligned on both sides of the channel 13 and the measuring electrodes 34M of the two pairs of sensing units are also aligned on both sides of the channel 13.

[0070] The electrodes 34 may be used for Trans-Epithelial Electrical Resistance (TEER) measurements in order to assess the strength, permeability and integrity of the epithelial and endothelial cells. In particular, the confluence of the monolayer cells 70 may be tracked and monitored in real-time as the TEER measurement will rise as the gaps in the monolayer cells 70 close. During a TEER measurement, a small voltage is appliedbetween one excitation electrode 34E and the other excitation electrode 34E. For example, the voltage is ranging from O.lmV to 150mV, preferably from 10 mV to 80 mV. Two measuring electrodes 34M measure how much current passes through the cellular layer 70. The current is thus dependent on the resistance caused by the cellular layer 70. The current is preferably alternating current. One could also measure TEER by applying a current on the electrodes 34E and measuring the resulting voltage between the measuring electrodes 34M.

[0071] The measurements may be performed at different frequencies. Low frequencies are highly sensitive to the intercellular barrier formed by tight junctions. The threshold frequency in the mid-frequency range describes the paracellular barrier formed by cell membranes. They are thus sensitive to the type of cells and the topology of the barrier. On the other hand, measurements at higher frequency can be used to assess the cell culture media (temperature, pH, ionic content).

[0072] The preferred configuration for performing TEER measurements is the improved embodiment as represented in figure 1.

[0073] Thanks to the deformability of the gasket layer 12, the carrier film 32, the electrodes 34 and their connecting leads 36 are squeezed between layers without changing the overall thickness of the multilayer stack 10. Therefore, the sensor 30 and the multilayer stack 10 are tight sealed.

[0074] In order to increase the sealing efficiency, the carrier film 32 and the electrodes 34 have a small thickness as compared to the thickness of the multilayer stack 10.

[0075] For example, the carrier film 32 has a thickness ranging from 1 pm to 100 pm, preferably ranging from 20 pm to 80 pm, even more preferably ranging from 40 pm to 60 pm. The carrier film 32 may be a biocompatible film such as a polyimide film. The carrier film 32 may comprise an adhesive coating on one or both sides. This is advantageous because this allows a precise positioning therefore reducing the time needed to assemble the multilayer stack 10.

[0076] The electrodes 34 may have a thickness ranging from 10 nm and 500 nm, preferably ranging from 90 nm and 200 nm, even more preferably ranging from 120 nm and 170 nm. The connecting leads 36 preferably have the same thickness, thus defining the thickness of the sensing unit. To reach such a low thickness, the electrodes 34 and the connecting leads 36 may be deposited on the carrier film 32, for example by printing or sputter coating.

[0077] The upper or lower support (21, 23) may also comprise additional sensors 60 as represented in figure 2A. For example, the upper support 21 may comprise an optical sensor that optically measures a specific substance. The optical sensor 60 is for example an optode comprising a chemical transducer.

[0078] The cell culture device may further comprise a base 40. The base 40 is placed below the lower support 23 to provide additional support and a better alignment of the layers of the multilayer stack and the upper and lower support. Moreover, the base 40 may comprise spring loaded pogo pins configured to apply a pressure on the extremity of the connecting leads 36 extending outside the multilayer stack 10 in order to increase the efficiency of the electrical connection with the measuring unit.

[0079] The cell culture device may further comprise a fastener configured to apply pressure on the multilayer stack 10.

[0080] For example, the fastener may be a set of screws fastening the upper support 21 and the lower support 23 and optionally the base 40 and applying a force aiming to bring the upper support 21 and the lower support 23 and optionally the base 40 closer together.

[0081] In another embodiment, the fastener is a lever vice. The lever vice may be configured to evenly distribute a pressure along the upper support 21 and the lower support 23. To do so, the fastener comprises an upper pressing element 51 configured to be uniformly disposed on the upper support 21 and a lower pressing element 53 configured to be uniformly disposed under the lower support 23 and optionally the base 40. The lever vice is configured to apply a force on each of the pressing elements (51, 53) in order to bring the upper support 21 and the lower support 23 and optionally the base 40 closer together. The pressure may be evenly distributed if, for example, each ofthe pressing element (51, 53) is disposed all along the circumference of the upper support 21 and the lower support 23. The lever vice may be metallic to increase the robustness compared to plastic clamp. The lower pressing element 53 may comprise alignment pins 23a, preferably six alignment pins 23a, extending in the vertical direction. In this embodiment, each of the layers (11, 12) of the multilayer stack 10, the lower support 23 and the upper support 21 comprises openings 80 cooperating with the alignment pins 23a. As shown in figure 1, the openings 80 of the lower support 23 may comprise a wall extending along the vertical direction thereby forming the alignment pins 23a cooperating with the openings 80 of the layers (11, 12) of the multilayer stack 10 and the upper support 21.

[0082] The fastening advantageously allows to provide a more uniform pressure inside the cell culture device over the time.

[0083] Moreover, the upper support 21 and the lower support 23 compress the multilayer stack 10. This allows to improve the sealing thanks to the deformability of the gasket layers 12 in the direction of the compression. The gasket layers 12 therefore enclose the sensor 30.

[0084] The invention also relates to a cell culture system comprising: the cell culture device described above, a pump fluidly connected to the inlet 13a and outlet 13b of each channel 13 for providing a cell culture medium to the channels 13; a measuring unit configured to receive signals from the electrodes 34.

[0085] The pump is for example connected to the inlet and outlet ports 21a of the upper support 21. The pump may be a peristatic pump. For example, the pump is configured to set a flow rate ranging from IpL / min to 30pL / min.

[0086] The cell culture medium may comprise nutrients. The cell culture medium may also comprise at least one active principle for which the interaction with the cultured cells 70 will be analyzed. The cell culture medium may also comprise oxygen.

[0087] The cell culture system may further comprise a bubble trap by which aqueous liquid is retained and bubbles are expelled through a microporous hydrophobic membrane, for example Polytetrafluoroethylene (PTFE) membrane. This ultimately results in a bubble free liquid coming out of the bubble trap. For example, the bubble trap is the Elveflow™ autoclavable bubble trap for microfluidics.

[0088] The cell culture system may further comprise a monitoring device configured to monitor at least one cell culture parameter. Moreover, the monitoring device may be configured to provide instructions to the pump for adjusting the fluid supply according to the measured cell culture parameter(s).

[0089] The invention also relates to a method of monitoring a cell culture. The method comprises the steps of: providing the cell culture device in any embodiments described hereabove; establishing a cell culture in at least one of the channels 13, the electrodes 34 being aligned with said channel 13; performing an electrical measurement with the sensor 30.EXAMPLES

[0090] The present invention is further illustrated by the following examples.Example 1 : Method for manufacturing the sensor

[0091] This example describes how to manufacture a sensor 30 being thin enough to provide thigh sealing with a gasket layer 12. The method is represented in figure 5.

[0092] First (step SI), a 55 pm thick adhesive polyimide film 32 is applied to a low adhesion layer such as a fluorinated foil carrier 31 (SILFLU S 50 M 1R88OO1 CLEAR, Siliconature, Italy) comprising alignment marks 31a and peeling cuts 31b on its surface. The alignment marks 31a allow to perform a better alignment of the electrodes 34 whereas the peeling cuts 31b allows for an easier peeling of the polyimide film 32 whenthe sensor 30 is manufactured. The polyimide film 32 may have an optimized metal adhesion in order to allow the polyimide film 32 to be peeled off from the fluorinated foil carrier 31 without degrading the conductivity of the thin-film electrodes 34.

[0093] Optionally (step S2), the fluorinated foil carrier 32 is attached to a rigid 700 pm thick glass wafer 33 to keep the polyimide film 32 flat during processing.

[0094] After wiping the polyimide film 32 with acetone and an acetone ultrasound bath during 3 minutes, followed by an isopropanol bath, the fluorinated foil carrier 31 with the polyimide film 32 is cleaned with a 1000 W O2 plasma during 1 minute.

[0095] To pattern the sensing units a standard lift-off process is used. A photoresist mask 35 (MICROPOSIT S 1813, micro resist technology GmbH, Germany) is applied on the polyimide film 32 and spun at 1700 rpm, resulting in a 1.7 pm thick layer (step S3).

[0096] After soft baking the photoresist mask 35 at 100°C for 2 minutes, the polyimide film 32 is exposed to the template of the electrode shape for 7 seconds with UV-light. Followed by image reversal (YES-58TA-E, Yield Engineering Systems, USA), and developing the photoresist mask 35 (Microposit™ Developer 351, micro resist technology GmbH, Germany) thereby forming trenches in the polyimide film 32 (step S4).

[0097] The trenches are then cleaned up with a descum program in the plasma oven, at 200 W N2 / O2 plasma for 2 minutes (step S5).

[0098] To prepare the surface for the sensing units deposition, the polyimide film 32 is treated with Argon and O2 plasma for 3 minutes and 5 minutes at 50 cm3 / m with 50 W (PE- 100, Plasma Etch Inc., USA) and placed in a table-top sputter (Q300TD, Quorum Technologies Ltd, UK) (step S6).

[0099] A 3 nm Tantalum (Ta) adhesion layer is deposited followed by a 150 nm Platinum (Pt) layer 34 (step S7). The photoresist mask (step S8) is then lifted off with, optionally, a 5 minutes acetone ultrasound bath followed by a 1 minute isopropanol bath.

[0100] In the final step the polyimide film 32 is cut to shape on the cutter plotter, using the alignment marks 31a of the fluorinated foil carrier 31 (step S9), and is then ready to be applied and sterilized (step S10).Example characterization of the electrodes

[0101] This example is based on the use of the basic embodiment of the cell culture device comprising three chambers. The device comprises four Platinum sensing units. The example is based on TEER measurements. TEER values (in Ohm Q) may be normalized to Q.cm2(Ohm multiplied by squared centimeter) in order to give a value of the barrier integrity that is not geometry dependent. A geometrical correction factor which is dependent on the TEER value is applied for proper normalization. The correction factor was calculated by finite element method (FEM) simulations.

[0102] First, Caco-2 cells (cancerous cell line) 70 were seeded in the first intermediate channel 15b on a collagen-coated 14 semi-permeable layer 11, which was primed with media.

[0103] Three hours post-seeding, the cells 70 were subjected to flow in the lower and upper channels (15a, 15d). The cultured cells 70 thus form a barrier in the form of a monolayer.

[0104] The cell barrier is then disrupted by calcium depletion in the cell media. After 24 hours of barrier disruption, fresh cell media was added to the lower channel 15a, the first intermediate channel 15b and the second intermediate channel 15c to allow cell barrier recovery.

[0105] Throughout the entire experiment, cellular impedance in Ohm (Q) (vertical axis of figures 6-8) was measured over a frequency range of 1-106Hz (horizontal axis of figures 6-8; logarithmic scale). The impedance was recorded on day 0 (DO; solid line), day 1 (DI; dot-dash line), day 2 (D2; dotted line), day 4 (D4; short dashed line), day 8 (D8; long dashed line) and day 12 (D12; dash-dot-dot line) of the culture (figure 6), andthe recorded data was analyzed before (BCa; solid line) and 1 hour (ACa-1; short dashdot line), 2 hours (ACa-2; dotted line), 3 hours (ACa-3; short dashed line), 6 hours (ACa- 6; long dashed line), 12 hours (ACa-12; dash-dot-dot line) and 24 hours (ACa-24; long dash-dot line) after Calcium depletion in the cell media (figure 7), as well as before (BCa; solid line) and 1 hour (ACa-1; dot-dash line), 2 hours (ACa-2; dotted line), 3 hours (ACa- 3; dashed line) after media refresh in the cell culture device (figure 8).

[0106] The results of figure 6 indicate an increase in impedance in the low frequency range between day 2 (D2) and day 12 (D12) after cell seeding in the cell culture device. The increase in impedance in the low frequency range is characteristic of the formation of a tight cellular barrier.

[0107] On day 12 after seeding, figure 7 shows that the impedance decreased after 2 hours (ACa-2) of calcium removal (BCa). However, figure 8 shows that upon replenishing the cell media with calcium-containing media, the cell function recovers after 2 hours (ACa-2) of calcium switch (BCa).

[0108] Contrarily to known devices, after the 12 days of experience, no leakage has been detected and the pressure was kept constant over the 12 days thanks to the device of the invention.Example 3: Cells co-culture

[0109] This example describes the protocol used to co-culture cells 70 in the cell culture device of the improved embodiment comprising four chambers as represented in figure 2B.

[0110] First, on day 1, the semi-permeable layers 11 are coated with collagen or mucin 14.

[0111] On day 2, the medium is prepared and transferred in glass bottles. The tubing lines are also prepared to connect the pump with the inlet 13a and outlet 13b of the channels 13.

[0112] On day 3, the cell culture device is assembled.

[0113] On day 4, the tubbing lines are connected to the device. Epithelial cells 70 are seeded in the first intermediate channel 15b. The upper channel 15d is filled with nitrogen. A TEER measurement was performed through the first intermediate channel 15b to determine the cell barrier integrity.

[0114] On day 8, bacteria are precultured outside the device.

[0115] On day 9, immune cells are prepared outside the device.

[0116] On day 10, the precultured bacteria is introduced in the second intermediate channel 15c and immune cells are deposited in the lower channel 15a.

[0117] TEER measurements are performed regularly so that to evaluate the Epithelial cell barrier integrity. Thanks to the device of the invention, measurements are only performed through the first intermediate channel 15b. The signal from the electrodes is thus not scrambled by the presence of the bacteria and immune cells in the other channels.

[0118] Moreover, during the overall measurements, the device does not suffer from leakages which would be the case if the electrodes of the known devices are inserted between the channels.NUMERICAL REFERENCES10: multilayer stack / / 11: semi-permeable layers / / 12: gasket layers / / 12a: communication hole / / 13: channel / / 13a: inlet / / 13b: outlet / / 13c: inlet and outlet duct / / 14: coating / / 15a: lower channel / / 15b: (first) intermediate channel / / 15c: second intermediate channel / / 15d: upper channel / / 21: upper support / / 21a: inlet and outlet ports / / 23: lower support / / 23a: alignment pins / / 30: sensor / / 31: fluorinated foil carrier / / 31a: alignment marks / / 31b: peeling cuts / / 32: carrier film / / 33: glass wafer / / 34: electrode / / 34E: exciting electrode / / 34M: measuring electrode / / 35: photoresist mask / / 36: connecting lead / / 37: connecting pad / / 40: base / / 51: upper pressing element / / 53:lower pressing element / / 60: additional sensor / / 70: cell / / 80: openings / / SI: application of adhesive polyimide film / / S2: attachment to glass wafer / / S3: application of photoresist mask / / S4: forming trenches / / S5: cleaning trenches / / S6: surface preparation / / S7: electrode deposition / / S8: lift off / / S9: cutting / / S10: ready / / DO: impedance on day 0 / / DI: impedance on day 1 / / D2: impedance on day 2 / / D4: impedance on day 4 / / D8: impedance on day 8 / / D12: impedance on day 12 / / BCa: Before calcium switch / / ACa- 1: 1 hour after Calcium switch / / ACa-2: 2 hours after Calcium switch / / ACa-3: 3 hours after Calcium switch / / ACa-6: 6 hours after Calcium switch / / ACa-12: 12 hours after Calcium switch / / ACa-24: 24 hours after Calcium switch

Claims

CLAIMS1. A cell culture device comprising: a multilayer stack (10) comprising at least five layers wherein: o at least three of the layers are gasket layers (12), each gasket layer (12) comprising at least one channel (13), each channel (13) comprising an inlet (13a) and an outlet (13b); and o at least two of the layers are semi-permeable layers (11); wherein the gasket layers (12) and the semi-permeable layers (11) are alternating; and wherein neighboring gasket layers (12) are superposed so that at least part of the channels (13) are aligned; an upper support (21) and a lower support (23), the multilayer stack (10) being disposed between the upper support (21) and the lower support (23); and a sensor (30) comprising at least four sensing units, each sensing unit being on a carrier film (32), each sensing unit comprising an electrode (34) connected to a connecting lead (36), each carrier film (32) being disposed on one of the layers (11, 12) of the multilayer stack (10), the electrodes (34) being aligned with the channel (13) of at least one of the gasket layers (12), wherein the sensor (30) and the multilayer stack (10) are tight sealed.

2. The cell culture device according to claim 1 wherein the gasket layer (12) is elastomeric.

3. The cell culture device according to claim 1 or 2 wherein the carrier film (32) is a polyimide film, preferably an adhesive polyimide film.

4. The cell culture device according to any one of claims 1 to 3 wherein the carrier film (32) has a thickness ranging from 1 m to 100 pm.

5. The cell culture device according to any one of claims 1 to 4 wherein the sensing units have a thickness ranging from 10 nm to 500 nm.

6. The cell culture device according to any one of claims 1 to 5 wherein the sensing units form a pair of sensing units, the electrodes (34) of a pair of sensing units being opposite on either side of one of the channels (13).

7. The cell culture device according to any one of claims 1 to 6 wherein the sensing units are deposited on the carrier film (32).

8. The cell culture device according to any one of claims 1 to 7 further comprising at least one fastener configured to apply pressure on the multilayer stack (10).

9. The cell culture device according to any one of claims 1 to 8 wherein each channel (13) comprises an inlet part comprising the inlet (13a), an outlet part comprising the outlet (13b) and a linear part between the inlet part and the outlet part.

10. The cell culture device according to any one of claims 1 to 8 wherein each channel (13) comprises a circular part between the inlet (13a) and the outlet (13b).

11. The cell culture device according to any one of claims 1 to 10 further comprising a base (40), the lower support (23) lying on the base (40).

12. A cell culture system comprising: the cell culture device according to claim 1 to 11 ; a pump fluidly connected to the inlet (13a) and outlet (13b) of each channel (13) for providing a cell culture medium to the channels (13); a measuring unit configured to receive signals from the electrodes (34).

13. The cell culture system according to claim 12 further comprising a bubble trap.

14. A method of monitoring a cell culture, the method comprising the steps of: providing any of the cell culture device of claims 1 to 11; establishing a cell culture in at least one of the channels (13), the electrodes (34) being aligned with said channel (13); - performing an electrical measurement with the sensor (30).

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