Patent Application
20220187276
The present invention relates to the field of micro-fluidic devices and the measurement of cell impedance and transepithelial electrical resistance (TEER) of cells and cell layers.
Monolayers of epithelial and endothelial cells form barriers within animals, in particular within humans, and regulate the free movement of molecules between different tissues and/or interstitial compartments. In many diseases, these barriers become compromised, and hence, measuring their permeability is of considerable interest to cell biologists.
Most epithelial and endothelial cell types can be cultured in vitro to form confluent monolayers where it is possible to measure the barrier function afforded by these cell layers. In addition, dynamic changes of the layers can be followed when the cellular environment is altered by exposure to substances (e.g. pharmaceutical compounds, toxic compounds) or physical changes such as mechanical or osmotic stress.
The barrier function (permeability) of cell monolayers can be measured using various methods. Some of these methods are based on the measurement of electrical resistance or impedance. The cells and cell layer to be examined is usually grown upon a solid substrate, upon a membrane filter or on a porous membrane.
Cells can be monitored upon a solid substrate with gold electrodes and typically performed using 96 well arrays. Alternatively, a cell layer may be applied on a membrane filter or a porous membrane as a substrate. The use of the aforementioned filters and membranes is advantageous since it simulates a more in vivo like situation where cells are effectively fed from both the apical and basal side. It is commonly observed that under these conditions cell layers achieve higher absolute barrier function.
Sbrana T. et al (Sensors and Actuators B: Chemical 223 (2015): 440-446) discloses the design and fabrication of a transepithelial electrical resistance (TEER)-bioreactor for the study of nanoparticle toxicity in intestinal epithelial cells.
US 2014/065660 A1 discloses a microfluidic biological barrier model, e.g. for measuring the transepithelial electrical resistance (TEER).
The measurement of electrical resistance and impedance requires the presence of electrodes close to the cells and cell layer. Current methods do not allow the measurement of transepithelial electrical resistance (TEER) and/or for determining the impedance in an accurate simulating an in vivo like situation where the cells are located on a porous membrane because the cells cannot be positioned close to the electrodes. Hence, it is an object of the present invention to provide methods and means allowing to determine the transepithelial electrical resistance (TEER) of a cell layer and/or the impedance of cells or a cell layer using porous membranes.
The present invention relates to a microfluidic device for determining the transepithelial electrical resistance (TEER) of a cell layer or a cell assembly and/or for determining the impedance of cells, a cell layer or a cell assembly, said device comprising at least one micro-channel comprising at least a lower and an upper compartment separated by at least one porous membrane and optionally an inner compartment, the lower compartment comprising a bottom wall and side walls, the upper compartment comprising an upper wall and side walls, the bottom and upper wall, the side walls and the at least one porous membrane defining compartment volumes, wherein at least one porous membrane comprises on its surface at least one electrode.
It turned surprisingly out that with the method of the present invention for producing a porous membrane comprising an electrode on its surface it is possible to apply electrodes in any design (e.g. interdigitated, strip or disc electrode) on porous and flexible membranes. This allows to manufacture microfluidic devices as defined above comprising electrodes on such porous and flexible membranes which are not supported by a stiff or inflexible solid support. The presence of electrodes on porous membranes makes it possible to determine the transepithelial electrical resistance (TEER) of a cell layer or a cell assembly, preferably hydrogel-free or hydrogel-containing three-dimensional cell assemblies, directly and more precisely because the electrodes on the porous membrane can be positioned directly in the vicinity of or beneath a cell layer.
A further advantage of the microfluidic device of the present invention is the possibility to perform TEER measurements without the influence of a porous membrane since the electrodes are positioned on a porous membrane in direct contact with a cell layer or cell assembly. Furthermore, the microfluidic device of the present invention allows to measure the cumulative TEER of cell layers and hydrogel-free or hydrogel-containing three-dimensional cell assemblys positioned on both sides of a porous membrane. Devices and methods known in the art require to perform two independent measurements.
Hence, another aspect of the present invention relates to a method for determining the transepithelial electrical resistance (TEER) of a cell layer comprising the steps of
The device of the present invention can also be used to determine the impedance of cells or cell layers by applying alternating current to the device comprising cells and/or cell layers being present on the porous membrane and/or any wall of the compartments of the microfluidic channel.
Thus, a further aspect of the present invention relates to a method for determining the impedance of cells, a cell layer or cell assembly comprising the steps of
The provision of electrodes on porous membranes, in particular on flexible porous membranes, as used in the devices of the present invention cannot be achieved using methods in the art.
Thus, another aspect of the present invention relates to a method for producing a porous membrane comprising an electrode on its surface comprising the steps of
Yet, another aspect of the present invention relates to a porous membrane obtainable by a method according to the present invention.
Another aspect of the present invention relates to a device as defined herein comprising a porous membrane obtainable by a method of the present invention.
The methods and devices described herein allow to study cell barriers using electrical impedance spectros-copy and/or TEER. The combination of thin film electrodes of any architecture located on porous, free-standing and flexible polymer membranes allows for the first time versatile interconnection of multiple electrodes in a micro-fluidic device of the present invention. This means that, compared to conventional TEER and impedance strategies, where just a single barrier plane can be tested, multiple cell barriers can be probed within a single multi-layered device using a variety of electrode designs including disc, band and interdigitated metal thin film electrodes. The microfluidic device of the present invention allows to correlate cell surface coverage using membrane-integrated metal thin film electrodes (2-point impedance measurement setup) with barrier integrity and tightness based on Trans-cellular resistance measurements (TEER; 4-point measurement setup).
The present invention in based on the combination of well-known electro-analytical biosensing strategies (electrical impedance spectroscopy and TEER) for cell and cell barriers analysis, thus creating synergetic effects that compensate for the individual limitations of both analytical methods. As a consequence, more complex biological structures comprising of multiple physiological cell layers can be investigated using a single method and a single microfluidic device.
As mentioned above the present invention allows for the first time to solve the problem of produce reliable structured metal films of a defined geometry (i.e. electrodes) down to 2.5 μm or less on porous, flexible polymeric membranes by using a simplified method that allows the fabrication of even highly interdigitated metal thin film electrodes on even 10 μm or less thick flexible porous and free-standing polymeric membranes.
The microfluidic device of the present invention may have any architecture provided that the device comprises at least one, preferably at least two, more preferably at least three, more preferably at least three, microchannels as defined herein. The at least one microchannel comprises two or more compartments, a lower and an upper compartment and optionally an inner compartment. These at least two compartments may have different inlets and different outlets so that both compartments may not be fluidly connected to each other. However, it is possible that one or more microchannels of the microfluidic device of the present invention have the same inlets and/or outlets.
The at least one microchannel comprises two compartments which are separated by a porous membrane comprising at least one electrode on its surface. The compartments of the at least one microchannel are confined by side walls and by a bottom or upper wall.
The device of the present invention can be used to determine the TEER of a cell layer or a cell assembly and/or for determining the impedance of cells, cell layer or a cell assembly. The cells may be eukaryotic cells, in particular mammalian cells. The cells may be epithelial or endothelial cells or stem cells capable to be differentiated into epithelial or endothelial cells.
The cell assembly used in the methods of the present invention may be a cell aggregate or any other combination of cells including hydrogel-based assemblies (e.g. cell mono- and co-cultures embedded in synthetic and/or natural hydrogels) as well as cellular self-assembly (e.g. spheroids). Hydrogels which can be used to embed cells include extracellular matrix extracts (Matrigel, Geltrex), fibrin hydrogels, silk hydrogels, collagen hydrogels, gelatin hydrogels, hydrogels from alginic acid (alginate) or composites thereof. Furthermore, synthetic hydrogels like dextran (e.g. PEG-dextran) can also be used.
According to a preferred embodiment of the present invention at least two porous membranes and side walls define at least one inner compartment volume being positioned between the lower and upper compartment.
The microchannel of the device of the present invention may comprise at least one inner compartment which volume is defined by two porous membranes and side walls of the microchannel.
According to a further preferred embodiment of the present invention at least one electrode on at least one porous membrane is facing the lower and/or upper compartment and/or at least one inner compartment.
The porous membrane within the at least one micro-channel of the device of the present invention may comprise at least one electrode on one or both sides of said membrane. However, it is preferred that only one side of the porous membrane comprises electrodes on its surface.
According to another preferred embodiment of the present invention the bottom and/or upper wall and/or at least one of the side walls of one or more compartments comprises at least one electrode on its surface, wherein it is particularly preferred that one or more compartments comprise on the bottom and/or upper wall (not the side walls) at least one electrode.
The porous membrane separating the compartments of the at least one microchannel comprise at least one electrode positioned on the surface of said porous membrane. However, also the upper wall, the bottom wall and/or the one or more sidewalls of the compartment may comprise at least one electrode positioned on their surface. This is particularly advantageous because it allows to determine the TEER of a cell layer by applying an electric current between the at least one electrode on the porous membrane covered by said cell layer and at least one electrode on the upper or bottom wall or side wall in a compartment resulting in the order electrode-cell layer-electrode.
According to a further preferred embodiment of the present invention the porous membrane and the bottom and/or upper wall and/or at least one of the side walls of one or more compartments comprise at least two electrodes on their surface.
The presence of at least two electrodes on the surface of the aforementioned elements of the at least one microchannel allows measuring impedance between the at least two electrodes whose electron flow can be impeded by the presence of cells or a cell layer on said surfaces. Furthermore, the provision of more than two electrodes on the surface of the porous membrane and the bottom and/or upper wall and/or at least one of the side walls of the one or more compartments increases the reliability of the TEER measurements.
According to a preferred embodiment of the present invention the at least one electrode on the surface of the at least one porous membrane is positioned substantially opposite to the at least one electrode on the surface a second porous membrane and/or of the lower and/or upper wall of one or more compartments.
It is advantageous to position the electrodes on the surface of the porous membrane substantially opposite to the at least one electrode on the surface of the lower first and/or upper wall of one or more compartments allowing a direct electron flow between at least two electrodes.
According to another preferred embodiment of the present invention the porous membrane comprises or consists of a polymer selected from the group consisting of polyesters, preferably polyethylene terephthalate, polystyrene, polycarbonate, polyether ether ketone (PEEK) or and thiol-ene polymers, preferably epoxy-containing thiol-ene polymers.
The aforementioned polymers can be used as part of or form themselves porous membranes which can be used in the device of the present invention.
The at least one porous membrane may have a thickness of 2 to 50 μm, preferably 5 to 20 μm, more preferably 5 to 15 μm, more preferably 8 to 12 μm, in particular approximately 10 μm.
According to a further embodiment of the present invention the side walls of the compartments of the at least one microchannel have a height of 1 to 1500 μm, preferably 5 to 1300 μm, more preferably 50 to 1250 μm, in particular approximately 1200 μm.
The electrodes comprise or consist preferably of a material selected from the group consisting of silver, gold, platinum, chromium, aluminium zinc oxide (AZO), indium tin oxide (ITO), iridium platinum, black platinum, titanium nitride and carbon.
The material used for manufacturing the electrodes on a surface within the microchannel of the device of the present invention should show a good electrical conductivity and be substantially inert against cell culture media and water used for cultivating cells or washing the microfluidic device of the present invention. Furthermore, the material shall be applicable on a surface using methods known in the art and shall be flexible enough to be resistant against movements of the porous membrane.
According to a preferred embodiment of the present invention the electrodes have a thickness of 10 to 1500 nm, preferably 15 to 250 nm, more preferably 50 to 75 nm, in particular approximately 70 nm.
According to a further preferred embodiment of the present invention at least two electrodes on said porous membrane, on said bottom wall, on said upper wall and on said side walls have a distance from each other ranging from 1 to 500 μm, preferably 10 to 250 μm, more preferably 10 to 100 μm, in particular approximately 15 μm.
The electrodes in the microfluidic device of the present invention may have different structures. Hence, according to preferred embodiment of the present invention the at least one porous membrane of the device of the present invention comprises on its surface at least two electrodes arranged as interdigitated electrodes having a plurality of digits forming a comb-like electrode pattern.
According to a preferred embodiment of the present invention
A device comprising such an arrangement can be used for determining the TEER of a cell layer or its impedance. In the course of a TEER measurement, the cell layer is preferably positioned between at least two electrodes wherein at least one electrode is positioned on the porous membrane and at least one electrode is positioned on the upper, bottom or a side wall of the compartment.
The surfaces of the microchannel, i.e. the porous membrane, the bottom wall, the upper wall and/or the side walls and optionally the electrodes, may be modified in a manner to prevent or support/allow the attachment of cells thereon. The provision of appropriate surface modifications allows to regulate the areas within the micro-channel of the device of the present invention which are covered by cells or cell layers. In order to support the attachment of cells on a surface within the microchannel of the device of the present invention said surface may be modified with natural ECM proteins (collagen, fibronectin, vitronectin, laminin, ECM protein motifs (RGD motif containing molecules, hydrogels and/or self-assembly monolayers), silanes (e.g. APTES for amino groups) or synthetic hydrogel layers (e.g. RDG-modified PEG).
Another aspect of the present invention relates to a method for determining the transepithelial electrical resistance (TEER) of a cell layer or a cell assembly comprising the steps of
The microfluidic device of the present invention can be used for determining the transepithelial electrical resistance (TEER) of one or more cell layers. In order to determine TEER cells are applied to at least one micro-channel of the device of the present invention. A cell layer shall be positioned preferably on the porous membrane comprising on its surface at least one electrode. The field strength applied during the measurements is typically less than typically applied for electroporating cells and determined empirically. Typically a voltage of 1 to 100 mV, preferably 2 to 90 mV, more preferably 5 to 80 mV, more preferably 10 to 70 mV, more preferably 30 to 60 mV, in particular 50 mV, is used.
A further aspect of the present invention relates to a method for determining the impedance of cells or a cell layer comprising the steps of
The device of the present invention can also be used for determining the impedance of cells or a cell layer. The cells may be positioned on a porous membrane, upper wall, bottom wall and/or one or more side walls.
According to a preferred embodiment of the present invention air is applied to the upper compartment comprising a cell layer on the porous membrane positioned to the upper compartment to remove substantially all culture medium present in said upper compartment creating an air-liquid interface and alternating current is applied at least two electrodes on said porous membrane.
According to another preferred embodiment of the present invention air is applied to the lower compartment comprising a cell layer on the porous membrane positioned to the upper membrane surface in the lower compartment to remove substantially all culture medium present in said lower compartment creating an air-liquid interface and alternating current is applied at least two electrodes on said porous membrane.
Advantage of these embodiments compared to conventional TEER analysis is that cell analysis can be performed in a humid gaseous environment in the absence of medium. Conventional TEER approaches can only be performed when medium is resupplied and the air-liquid interface is discontinued during cell analysis.
The air applied to one of the compartments of the device of the present invention allows to determine the influence of gases on the cells of a cell layer. Thus, the composition of the air applied to one of the two compartments can be varied by introducing other gases or by changing its composition.
According to a preferred embodiment of the present invention the impedance and/or cell resistance and/or capacitance at the air-liquid interface is measured without the presence of an electrolyte in the upper or lower compartment. This means that cell monitoring at an air-liquid interface can be tested without the need for resupplementation of culture medium, which is a manipulation of the culture condition during cell analysis
According to a further preferred embodiment of the present invention the air applied to the lower or upper compartment comprises 78% nitrogen, 21% oxygen, optionally the air applied to the compartments may contain carbon dioxide at 5% and nitrogen environmental conditions.
The alternating current is preferably applied to the electrodes of the device of the present invention at a frequency of 5 Hz to 5 MHz, preferably of 10 Hz to 2 Mhz.
The porous membrane comprising at least one electrode on its surface is produced using a method as described below. This method allows to cover porous and flexible membranes with electrodes of different structures.
Thus, another aspect of the present invention relates to a method for producing a porous membrane comprising an electrode on its surface comprising the steps of
The method of the present invention is particularly advantageous because it allows applying electrodes on porous, preferably flexible, membranes. In particular the final step of the method wherein water or an aqueous solution instead of on organic solvent like acetone is used to release the membrane from the solid support allows manufacturing electrodes on porous membranes. The use of water, preferably deionized water, and aqueous solutions prevents the deterioration of the porous membranes and the electrodes during their manufacturing process.
“Aqueous solution”, as used in the method of the present invention, may be any solution comprising more than 90 wt %, preferably more than 95 wt %, more preferably more than 98 wt %, water.
The solid support used to manufacture the porous membrane of the invention can be of any material. However, it is particularly preferred that the solid support comprises or consists of a material selected from the group consisting of glass, silicon, silicon nitride, silicon dioxide, zirconium dioxide.
According to a preferred embodiment of the present invention the water-soluble synthetic polymer is selected from the group consisting of polyvinyl alcohol (PVA), poly acrylic acids (PAA) or dextran.
According to a further preferred embodiment of the present invention 2 to 10 wt %, preferably 3 to 5 wt %, more preferably approximately 4 wt %, polyvinyl alcohol with a molecular weight of 5,000 to 30,000, preferably 13,000 to 23,000, in deionised H2O is deposited on the solid support, so that surface is covered, the height of the deposited layer is defined by spin coating with 800 rpm for 30 s, in optional step b).
According to a preferred embodiment of the present invention the solid support is baked after step b), d), e) and/or g) by applying temperatures up to 180° C. for up to 300 s.
According to another preferred embodiment of the present invention the water-insoluble synthetic polymer is selected from the group consisting of poly(methyl methacrylate), polystyrene, cyclic olefins (topas COC, zeonor COP), thiol-enes or thiol-enes-epoxies is deposited directly on said solid support. The water insoluble polymer is comprised within an appropriate solvent in an amount of 0.1 to 30% w/v, preferably 1 to 25% w/v.
According to a preferred embodiment of the present invention the optional layer comprising a polydimethyl-glutarimide based resist, preferably LOR3A or LOR3B, is applied on the solid support using spin deposition, preferably spin deposition at 500 to 2000 rpm for 15 to 60 s.
The photoresist is preferably selected from the group consisting of positive, negative or image reversal resists (e.g.:AZ5214E—novolak resin and naphthoquinone diazide based).
According to a preferred embodiment of the present invention the photoresist is deposited on the solid support of step c) or d) with a thickness of about 1 to 2.5 μm, preferably about 1.2 to 2 μm, more preferably about 1.5 to 1.7 μm, more preferably about 1,62 μm, preferably by spin deposition at 1,000 to 5,000 rpm, preferably at 2,000 to 4,000 rpm, more preferably at approx. 3,000 rpm, for preferably 15 to 60 s, preferably approx. 30 s.
According to a further preferred embodiment of the present invention the solid support of step f) is exposed to ultraviolet radiation at a dose of 5 to 500 mJ/cm2, preferably of 10 to 400 mJ/cm2, more preferably of 15 to 300 mJ/cm2, more preferably of approx. 20 to 250 mJ/cm2.
According to a further preferred embodiment of the present invention the developer applied to the solid support of step g) is selected from the group consisting of Tetramethylammonium hydroxide (TMAH) and TMAH based developers (e.g.: AZ726MIF—containing 2.38% TMAH).
According to another preferred embodiment of the present invention the electrode material is selected from the group consisting of silver, gold, platinum, chromium, aluminium zinc oxide (AZO), indium tin oxide (ITO), iridium platinum, black platinum, titanium nitride and carbon.
According to a preferred embodiment of the present invention the electrode material is applied on the solid support of step h) or i) by sputtering, thermal evaporation, e-beam evaporation, screen printing or inkjet printing of conductive metal- or carbon-containing inks.
Another aspect of the present invention relates to a porous membrane obtainable by a method according to the present invention.
A further aspect of the present invention relates to a device according to the present invention comprising a porous membrane according to the present invention.
The present invention is further illustrated in the following figures, embodiments and examples without being restricted thereto.
First the Polyvinyl alcohol (PVA) used as a release layer needs to be prepared. In order to allow for fast dissolution of the PVA only low molecular weight PVA (13000-23000 Da) should be used. 4 g of PVA are dissolved in 100 ml deionized H2O (diH2O) and stirred at 70° C. (covered with aluminum foil to prevent evaporation of water) until the PVA is fully dissolved, once the PVA is dissolved the solution is filtered through a syringe filter (22 μm) to remove particles. Once the PVA is at room temperature it can be spin coated onto the carrier substrate (e.g. glass). The glass carrier substrate (Schott D263T eco) is cleaned using Acetone and Isopropyl alcohol and then placed on a hot plate set to 100° C. to evaporate any remaining solvent. After cleaning the glass substrate is treated with O2 plasma (300 W; 0.7 Torr; 45 seconds) to allow for easier spreading of the PVA release layer. The plasma treated glass substrates are transferred to a spin coater and the PVA is spread using a transfer pipette or syringe and then spun at 800 rpm for 30 seconds. After spin coating of the PVA release layer, pre cut PET Membrane pieces (slightly larger than the carrier substrate) are carefully placed on the carrier substrate, trying to avoid wrinkles (it helps to bend the membrane a little)—once the membrane has been in contact with the PVA it shouldn't be moved anymore! The membrane should be placed onto the carrier while the PVA is still wet. In order to dry the PVA the substrates with the membrane attached are placed on a hotplate and temperature is ramped to 150° C. (the LOR3A resist needs to be baked at this temperature). If no hotplate with a ramping function is available, or the ramping is too time consuming—the samples can be also baked gradually using hotplate set to 70° C., 100° C., 120° and 150° C. for 180 seconds each. If the samples are baked too fast the evaporating water will cause wrinkles on the membrane. After dehydration the samples are cooled to room temperature, membrane pieces overlapping the carrier substrate are cut away using a scalpel and LOR3A resist is spin coated at 1000 rpm for 30 s and then soft baked at 150° C. for 180 s—again the temperature should be ramped (or as mentioned above baked gradually). Once the LOR3A has been soft baked, AZ5214E Resist is spin coated at 3000 rpm for 30 s and then soft baked at 100° C. for 30 s. Using a photo mask, the desired electrode geometry is transferred to the sample by UV light (365 nm) exposure with a dose of 40 mJ/cm2. After exposure the sample is baked at 120° C. for 70 s and then flood exposed (without photo mask) to a dose of 240 mJ/cm2. Next the sample is developed in AZ726MIF (TMAH based developer) for 120 s (usually AZ5214E needs to be developed for 60 s—but TMAH dissolves LOR3A and allows for an undercut of the actual photo resist), and then rinsed in diH2O. Before continuing the samples should be dried (e.g. with Nitrogen spray gun, overnight in a desiccator). Before depositing the metal layer, the samples are subjected to an Argon plasma (50 W RF; 10 sccm Ar; 60 s), thereby modifying the parts of the membrane not covered by photoresist. After Plasma treatment a gold layer of approximately 80 nm is deposited by sputtering (25 W, 2×60 s sputter duration, base pressure: 2e{circumflex over ( )}-5 mbar, working pressure 8e{circumflex over ( )}-3 mbar) or evaporation. The sputter power shouldn't exceed 25 W, otherwise the membrane might overheat, or the metal might crack or spall during lift-off because of strain/tensile forces. After sputtering the samples are soaked in N-methyl pyrrolidone or N-Ethyl pyrrolidone for 10 minutes and the sonicated at low power to remove the photo resist and non-patterned gold. The membrane can be released by soaking the sample in diH2O for 1 min and then carefully pulling it off with tweezers.
Using the process, gold electrodes can be deposited on porous membranes achieving a resolution down to 2.5 μm. This process can also be used to deposit other metals (e.g. copper, chromium, titanium), or combinations thereof. When depositing metal combinations using sputtering, only a low sputtering power should be used to avoid spalling or cracking of the metals. The PVA release layer allows for rapid detachment of the membrane from its carrier and aids further processing.
Membrane electrodes were fabricated according to the Methods mentioned above, the microfluidic device was built by sand-blasting the culture chambers into micro-scope slides, and attached to the membrane using ARCare 90445 double sided adhesive tape. Microscope slides with drilled media inlet and outlet ports attached with ARCare 90445 were used to seal the chambers. As controls, BeWO b30 cells were seeded similarly on corning trans-well inserts with 3 μm pores and analyzed with a STX2 EVOM2 voltohmmeter after 45 minutes of cool-down.
Bewo B30 cells were routinely cultured in Ham's/F12 Media supplemented with 10% FBS, 1% Penicillin/Streptomycin and incubated at 37° C. and 5% CO2. Cells were detached from culture vessels using trypsin, centrifuged and seeded at different densities (100k/cm2, 50k/cm2, 25k/cm2 and 12.5k/cm2) in culture media supplemented with 2% HEPES. Cells were propagated on the chip up to a duration of 5 days with daily medium exchange.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19168889.4 | Apr 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/060323 | 4/10/2020 | WO | 00 |
