NL2026235B1

NL2026235B1 - Organ on chip

Info

Publication number: NL2026235B1
Application number: NL2026235A
Authority: NL
Inventors: Gerjanne Bernadette Maria Bossink Elsbeth; Odijk Mathieu; Irene Segerink Segerink Loes
Original assignee: Univ Twente
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2022-04-08
Also published as: WO2022031173A1

Abstract

The invention provides a system (1000) comprising a microfiuidic device (1), wherein the microfiuidic device (1) comprises a first side (2), a second side (3), a device plane (6) between the first side (2) and the second side (3), a first channel (100) having a first channel inlet (101) and a first channel outlet (102), and a first side chamber (150) extending from the first channel (100) between the first channel inlet (101) and the first channel outlet (102) in a direction parallel to the device plane (6); wherein the first side chamber (150) is accessible via a first opening (151) either via the first side (2) or via the second side (3); and wherein the system (1000) further comprises a first electrode element (8) at least partly configured in the first opening (151) and the first side chamber (150).

Description

P1600148NL00 Organ on chip

FIELD OF THE INVENTION The invention relates to a system comprising a microfluidic device, to a method for fabricating a system comprising a microfluidic device, and to a method for analyzing biological cells and/or biological tissue.

BACKGROUND OF THE INVENTION Organ-on-chip devices and methods for performing transepithelial/transendothelial electrical resistance (TEER) measurements with such devices are known in the art. WO2018157073, for instance describes an organ-on-chip device for monitoring a biological function and including a membrane layer located at an interface region between a top microchannel and a microchannel. The membrane includes a first type of cells forming a barrier between the top microchannel and the bottom microchannel. The device further includes a top layer having a first plurality of transendothelial electrical resistance (TEER) measurement electrodes for enabling direct monitoring of cell function and electrical activity of the first type of cells on the membrane.

SUMMARY OF THE INVENTION Organ-on-chips may be defined as microfluidic cell culture devices that mimic organ-specific functions. These devices are a potential alternative to conventional animal and in-vitro models being able to emulate complex human physiology in a miniaturized and highly controlled environment with the capability to provide a better model for drug screening. Organ- on-chip devices are usually made from biologically inert polymers such as polydimethylsiloxane (PDMS) and may comprise microfluidic culture channels lined with organ-specific cell types. Typically, organ-on-chip devices comprise two (parallel) culture channels, especially separated by a porous membrane. By integrating a porous membrane between the two parallel channels, e.g. barrier tissues can be studied which are essential for maintaining homeostasis of the organs and regulating the transport of certain compounds. Assessment of barrier integrity in such in-vitro models provides valuable information for further clinical studies and barrier targeting drug development. A common method to access the barrier properties is to study the trans-epithelial/endothelial electrical resistance (TEER) of cell culture. TEER may provide continuous, non-invasive, and label-free monitoring of the tightness of cell-cell junctions by measuring electrical resistance across a cellular barrier.

Integrated TEER electrodes in a typical organ-on-chip device have been already reported. Using advanced microfabrication technologies, miniaturized sensors can be directly embedded in, or in close proximity to the cell culture channels. This may be accomplished by patterning electrodes on the top and bottom substrates of the channels. Although these methods provide stable and reliable TEER measurements, the electrodes often block the possibility for visual inspection of the cells at the site of the electrode. Proposed solutions to solve this issue suggest the use of very thin electrodes of gold or indium tin oxide. However, they all require the sputtering of electrodes, an expensive and cleanroom required process.

The barrier function in in Transwell models can be accessed with commercially available systems, such as the EVOM? Volt/Ohmmeter. This system has ‘chopstick’ type electrodes, which are inserted on both sides of the Transwell insert. The resistance of the path between the electrodes, also through the cell layer, is measured by applying a direct or alternating current. This ‘chopstick method’ is also applied for typical organ-on-chip devices, by placing the electrodes in the in- and outlets of the microfluidic channels. However, using this method raises concerns about the stability of the electrode position and the membrane area that is actually probed, making it very difficult to obtain reliable measurements.

Further, there appears to be a need for scaling up the number of microfluidic chambers in one device. This will allow performing parallel experiments and analysis of multiple conditions increasing the throughput per chip. There appears to be a need to study the TEER in an organ-on-chip device with a cleanroom free, accessible fabrication method, which furthermore allows parallelization and especially also the visual inspection of the cells in the culture device.

Hence, it is an aspect of the invention to provide an alternative organ-on-chip device, which preferably further at least partly obviates one or more of above-described drawbacks. It is especially an aspect of the invention to provide a system comprising a microfluidic device, especially an organ-on-chip device, which preferably further at least partly obviates one or more of above-described drawbacks. It is a further aspect of the invention to provide a method for fabricating such system comprising a microfluidic device, which preferably further at least partly obviates one or more of above-described drawbacks. It is a further aspect of the invention to provide an alternative method for determining a property of a biological cell with an electric signal-related measurement, especially by applying TEER measurements, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

In a first aspect, the invention provides a system comprising a microfluidic device (“device”). The microfluidic device especially comprises a first side, and a second side (especially configured opposite to the first side) (such as respectively a top side and a bottom side). Further, the microfluidic device may especially comprise a device plane. The device plane may be (configured) between the first side and the second side. Further, the microfluidic device especially comprises a first channel, and a first side chamber. The first side chamber is especially fluidly connected to the first channel. In further specific embodiments, the first channel has a first channel inlet (for providing a fluid in the first channel). The first channel may further have a first channel outlet (for releasing the fluid from the first channel). In embodiments, first channel inlet may (also) function as a (first channel) fluid outlet. In further embodiments, the first side chamber extends from the first channel. The first chamber especially extends from the first channel (at a location) between the first channel inlet (upstream of the first chamber) and the first channel outlet (downstream of the tirst chamber). The first side chamber especially extends from the first channel in a direction parallel to the device plane. In further specific embodiments, the microfluidic device comprises a first channel comprising (or with) a first side chamber extending from the first channel. Further, in embodiments, the first side chamber is accessible via a first opening, especially either via the first side or via the second side (of the device). In specific further embodiments, the first side chamber is (thus) accessible via the first opening from a (single) side selected from the first side and the second side. The first opening is further especially configured for receiving (at least part of) a first electrode element. The first opening may further be configured for arranging at least part of the first electrode element in the first side chamber. In further specific embodiments, the system further comprises the first electrode element, especially at least partly configured in the first opening and (in) the first side chamber.

In a further aspect, the invention provides a method for fabricating a system comprising a microfluidic device. By application of the method, especially embodiments of the system of the invention may be provided. The method may especially comprise a preparation stage and a connecting stage. In embodiments. the connecting stage comprises providing a first layer with a primary first layer face comprising a (continuous) (especially elongated) first recess, especially wherein the first recess comprises a first side extension,

especially one or more first side extensions. The first side extension(s) may extend away from the first recess, especially along the primary first layer face. In further embodiments, the connecting stage further comprises covering the first recess with a cover, especially wherein a first cover side closes the continuous first recess, and further especially to provide the microfluidic device comprising a first channel and a first side chamber (especially one or more first side chambers) extending from the first channel. Especially (therefore), the first side chamber(s) is (are) fluidly connected to the first channel. The first recess may especially define the first channel and the first side chamber(s). Further, the first recess especially comprises the first channel axis.

Further, in embodiments, the preparation stage comprises providing a first opening (through the first layer) from a secondary first layer face (opposite to the primary first face) to the primary first layer face (to the (continuous) first recess) at a location of the first side extension. In embodiments, the preparation stage comprises providing the first opening from the secondary first layer face to the first side extension. In further embodiments a first opening is provided from the secondary first layer face to each of the one or more first side extensions. In further (especially alternative) embodiments, the preparation stage comprises providing the first opening (through the cover) from the first cover side to a second cover side (especially located opposite to first cover side) at a location of the cover configured for covering the first side extension. The first opening may especially be configured for having the first opening from the first cover side to the first side extension when the first recess is covered by the cover. The first opening may in embodiments be provided before the connecting stage. In further embodiments, the first opening may (too) be provided during the connecting stage. Additionally, or alternatively, the first opening may be provided after the connecting stage. The opening may e.g., be punched or drilled through the device after the connecting stage.

Optionally before or after providing a first electrode element in the first opening remaining open connections to external of the device may be closed again, e.g. using glue or another filler material. Hence, in embodiments the first opening is provided before, during and/or after the connecting stage. In further embodiments, the preparation stage is applied before, during and/or after the connecting stage. Further, in specific embodiments, a first electrode element is at least partly configured in the first opening and the first side chamber. The first electrode element is especially fixed in the first opening. Fixing the electrode element in the opening may improve a reliability and/or reproducibility of the measurements. The first electrode element may, e.g., be glued in the first opening.

The system of the invention may be obtained using a cleanroom-free fabrication method for the integration of one or more electrodes/electrode elements. The system may allow real-time measuring of the barrier resistance in the microfluidic device (in an organ-on-chip device) by means of impedance spectroscopy. Embodiments, of the system may allow 5 multiplexing the electrodes and/or the fluid channel (or organ-on-chip) in one device. The device may comprise a plurality of electrodes / electrode sets along the channel of the organ- on-chip, allowing to measure at different sites of the channel. The system and the method may further enable the configuration of multiple (same or different) organ-on-chips in one device, all containing TEER electrodes. In further embodiments, the system may allow visible inspection of (cells layers in) the channel, especially wherein the line of sight is not blocked by an electrode. In embodiments multiple electrodes may be integrated at predetermined locations such that the paths between the cooperating electrodes is defined and constant (having a constant base resistance between the electrodes). In embodiments electrode elements may be removed and reinserted, to further control the location of the electrodes with respect to further electrodes and/or to the biological cell under investigation.

Hence, the invention provides in a specific embodiment, a system comprising a microfluidic device, wherein the microfluidic device comprises (i) a first side, (ii) a second side, (iii) a device plane between the first side and the second side, (iv) a first channel (especially comprising a first channel inlet and a first channel outlet), and (v) a first side chamber (fluidly connected to the first channel and) extending from the first channel (especially between the first channel inlet and the first channel outlet) in a direction parallel to the device plane; wherein the first side chamber is accessible via a first opening either via the first side or via the second side. In further specific embodiments, the system further comprises a first electrode element at least partly configured in the first opening and the first side chamber.

The device plane is essentially a virtual plane. In general, the device plane may be parallel to one or more of the first side and the second side. In embodiments, the first side and the second side may define a (device) body. Further, the device body may define the first plane and the second plane. Especially the device body comprises the first channel. Further, especially the device body may comprise the first layer.

The first channel may essentially have any arbitrarily shape. The first channel may be curved or may be straight. Essentially, the first channel is configured for hosting and/or transporting a fluid. The first channel is especially elongated. The first channel may especially have a straight configuration over at least part of the first channel. The first channel may have a (longitudinal) first channel axis. The first channel may further have a rectangular cross sectional shape (perpendicular to the first channel axis). The cross sectional shape may in further embodiments be square or circular. The cross sectional shape may be elliptical. The cross sectional shape may have any arbitrary shape. Especially, the cross sectional shape of the first channel is rectangular (including square). Further, a characteristic dimension of the first channel may in embodiments be in the millimeter range. The characteristic dimension of the first channel (such as a width of the channel for a channel having a rectangular cross section or a diameter for a circular channel) may in embodiments be selected in the range of 0.1-10 mm, especially 0.2-7 mm, such as 0.5-5 mm.

The first channel especially comprises a first extreme and a second extreme of the first channel. One of the extremes may e.g. comprise a first channel inlet (for providing a fluid in the first channel and optionally releasing a fluid from the first channel). The other extreme may be closed or may e.g. comprise a first channel outlet. The first channel extremes may in embodiments be arranged at an edge of the microfluidic device. For instance a first channel inlet may be arranged at a first edge and a first channel outlet may be arranged at a further especially opposite) edge, and especially the first channel may be a straight channel connecting the first channel inlet and the first channel outlet. In further embodiments, the extremes comprise one or more of the first channel inlet and the first channel outlet (when present) may be configured at the first side or the second side of the microfluidic device.

The (one or more) first side chamber(s) is (are) especially configured between the extremes of the first channel.

Further, a first channel wall may surround the first channel. The first channel may in embodiments comprise a circumferentially closed channel {closed by the first channel wall). Yet, in further embodiments, the first channel wall may only partly circumferentially close the first channel (in the first layer), and one or more further elements may circumferentially close the first channel. The first channel wall may in embodiments, e.g., be defined by the first recess and the cover may circumferentially close the first channel. In further embodiments, the first channel may be formed by a first elongated cavity or space between two parts of a channel wall and that e.g. is closed at a third side by a further element, defining a further part of the channel wall. The further element may for instance be a further layer, such as a polymeric layer or a glass layer that may connect the two parts of the channel wall.

The first channel wall may be transparent (for visible light) to allow visual inspection of the first channel or elements in the first channel. The first channel may e.g. be configured (as the recess) in the first layer and especially the first layer may define the first channel wall. Hence, especially in embodiments, the first layer may be transparent. The first layer (and/or first channel wall) may e.g. comprise a transparent material such as a (first) transparent polymeric material. Examples of transparent polymeric materials that may be used are e.g. polydimethylsiloxane (PDMS), thiol-ene polymers, polycarbonate (PC), polyethylene terephthalate (PET), polyethylene (PE), polyesters, polystyrene (PS), cyclic olefin copolymers, etc. The thiol-ene polymer may in embodiments comprise an off-stoichiometry thiol-ene (“OSTE”) polymer (comprising off-stoichiometry blends of thiols and allyls). Alternatively or additionally a polymer known under its brand name Flexdym may be used. Hence, in embodiments substantially all (transparent) material of the first layer may function as at least part of the first channel wall. The first layer may further comprise another transparent material, such as glass or a transparent ceramic. In embodiments, the first layer comprises a polymeric material with one or more cavities, wherein the one or more cavities are closed by a transparent further layer. The transparent further layer may close the cavities especially at the first side.

As described above, the first side chamber may extend from the first channel. In further embodiments, a plurality of first side chambers extend from the first channel (in a direction parallel to the device plane), see further below. Especially also a wall of the one or more first side chambers is transparent. Also, the first side chamber wall may be defined by the (transparent) first layer. The term “wall” especially relates to a wall at least partly enclosing the chamber. The wall may therefore include a side wall, or a ceiling (wall), or e.g. a floor (wall).

The first side chamber (especially all first side chambers, see below) especially extends from the first channel, especially in a direction transverse to, especially perpendicular to, the first channel axis. Further, the first side chamber(s) extend(s) in a direction parallel to the device plane. The first side chamber is essentially configured (relative to the first channel) for allowing controlled measuring/sensing of an electric signal (with the first electrode element) in the proximity of the first channel while minimizing any (physical) disturbing of the environment in the first channel. Moreover, the first opening is especially configured for arranging the first electrode element in the first side chamber such that the first electrode element substantially does not disturb the environment in the first channel. The first electrode element (when configured in the first opening) is especially configured not to (physically and/or visually) block the first channel.

In embodiments, the first electrode element is configured substantially entirely remote from the first channel. In further embodiments, a first portion of the electrode element configured in the first side chamber is larger than a second portion of the in the electrode element configured in the first channel. For instance 10% of the electrode element may be configured in the first side chamber and less than 10%, such as less than 5%, or less than 1% (including 0%) may be configured in the first channel.

These percentages are especially relative to any arbitrary quantity and may e.g. relate to a length of the electrode element or e.g. to a volume of the electrode element, or a (total) surface area of the electrode element.

Hence, in further embodiments 50% of the first electrode element may be configured in the first side chamber and less than 50 vol%, such as less than 40 vol%, especially less than 10 vol%, such as 1 vol% or even less may be configured in the first channel.

The electrode element may comprise one or more electrically conductive materials, e.g., a metal or a carbon.

In embodiments, the electrode element comprises a platinum wire, or a gold (plated) wire.

The electrode element may in further embodiments comprise a rod or a pin.

The electrode element may be part of a printed circuit board (PCB) (comprising at least part of the electrode element extending from the PCB.

The term electrode element may in embodiments relate to a probe.

The (first) electrode element may further at least comprise an electrode

Hence, especially, the first electrode element does not extend into the first channel (or in any other channel, see further below). Further, the first side chamber may be in direct physical contact with the first channel.

Yet, in further embodiments, a (first) side channel may physically and fluidly connect the first side chamber to the first channel.

In further embodiments, a side channel and a further volume may define the first side chamber.

The first side chamber may essentially be configured for providing a fluid connection to the first channel and allowing fluid to enter.

The first side chamber may be configured to prevent the generation of a so called “capillary valve” between the first channel and the first side chamber.

Especially a junction between the first side chamber and the first channel is gradual to prevent any gaseous bubbles) accumulation that may close of the chamber for a liquid present in the first channel.

The first side chamber may in embodiments have an at least partly tapered shape in a direction perpendicular to the first channel axis.

The tapered shape is especially broadest closest to the first channel axis and may become narrower further extending from the first channel.

The tapered shape may facilitate fluid entering the first side chamber.

The first side chamber may further have a small volume in relation to the first channel.

The first side chamber may in embodiments extend from the first channel over an extension length that is equal to or smaller than twice the characteristic dimension (or width) of the first channel, especially equal to or smaller than the characteristic dimension (width) of the first channel.

The size of the first side chamber, or the extension length may especially be based on a (minimum) size of the first electrode element.

In further embodiments, a (total) volume of the first side chamber is equal to or smaller than 10 mm’, such as equal to or smaller than 1 mm). The volume of the first side chamber may, e.g., be in the range of 0.001-10 pl.

The volume of the first side chamber is especially at least 0.001 pl, even more especially at least 0.01 ul, such as at least 0.05 pl.

Herein the term “first side chamber” may refer to a plurality of first side chambers.

In embodiments, e.g. the microfluidic device comprises the first channel and a plurality of first side chambers.

In embodiments, a plurality of first side chambers may extend from the first channel.

For instance, 2 first side chambers may extend from the first channel or 4 first side chambers, or at least 3, such as at least 6, or 12, or even more, see also below.

In embodiments, only 1 first side chamber may extend from the first channel.

In embodiments, a plurality of first side chambers is fluidly connected to the first channel.

The plurality of first side chambers may comprise a plurality of different side chambers, e.g. some having a different shape or not all being accessible via a first opening from the same side (selected from the first side and the second side). Yet, in specific embodiments, at least two of the plurality of first side chambers, especially substantially all of the first side chambers are similar (yet may e.g. be configured mirrored with respect to the first channel axis). As such, the plurality of first side chambers may all extend from the first channel in a direction parallel to the device plane.

The individual first side chambers may extend in opposite directions from the first channel.

Further, especially each individual first side chamber is fluidly connected to the first channel.

Especially each individual first side chamber is accessible via a respective (individual) first opening.

In further embodiments, each individual first side chamber is accessible via a respective first opening via a (single) side selected from the first side and the second side.

Hence, in embodiments, the microfluidic device comprises two or more (a plurality of) first side chambers, especially extending from the first channel in a direction parallel to the device plane.

Each first side chamber is especially fluidly connected to the first channel.

Each first channel may comprise a first channel inlet and a first channel outlet.

One or more of the first channel inlets may further be fluidly connected to each other at a location upstream of the (respective) first fluid inlets.

Likewise one or more of the first channel outlets may be fluidly connected to each other at a location downstream of the first fluid outlets.

The first fluid channels may, e.g., be configured as a branched channel system.

For instance, a first channel part (for feeding/providing the fluid) may branch into two further channel parts, fluidly connected to the first channel inlets of two first channels.

In further embodiments, at least one of the two further channel parts may also branch again in two channel parts, resulting in three or four branches of the channel system, that may e.g. each again being fluidly connected to three or four individual first fluid inlets of four respective first channel, et cetera, et cetera.

It will be understood that the first channel outlets may be arranged likewise (but in reverse order, i.e. not comprising branching channel parts but with combining channel parts).

The microfluidic device may comprise one or more cavities/passages or openings from either the first side or from the second side (in)to a respective first side chamber.

The term “first opening” may refer to such cavity/passage. The first opening may at least be part of such passage. The one or more first openings, and/or cavities/passages, are especially configured for receiving (at least part of) a first electrode element, especially to partly host the first electrode element in the first side chamber. Hence, in further embodiments, each first side chamber is accessible via a respective first opening via the first side or each first side chamber is accessible via a respective first opening via the second side. In alternative embodiments, a first subset of the first side chambers is accessible from the first side and the remainder of the first side chambers is accessible from the second side.

The configuration wherein each first side chamber is accessible via an individual opening via the first side or via the second side allows to configure a plurality of comparable first channels (each one fluidly connected to one or more individual first side chambers) in the microfluidic device in an advantageous way. The plurality of first channels may be configured in a (single) plane parallel to the device plane. In such configuration, an individual first electrode element may be (partly) configured (from the first side or from the second side) in any one of the first side chambers that is fluidly connected to one of the first channels and in its respective first opening without damaging or interfering with another first channel. In further embodiments all first side chambers are accessible from (or via) a single side selected from the first side and the second side. Such embodiment may advantageously be combined with embodiments wherein the first channel inlets and/or first channel outlets are configured at the (same) single side Hence, the term “first channel” may herein especially refer to a plurality of first channels, such as n first channel. The number of n may in embodiments be 2. The number n may also be at least 3, or 4, or 6, or 8, or e.g. 16, or even 32, or at least 64, such as at least 128. The number n may especially only be limited by the size of the first channel with respect to the size of the microfluidic device and may even be more than 200. Yet, in embodiments n is equal toor smaller than 128, such as equal to or smaller than 96, or equal to or smaller than 12.

In further embodiments, the device comprises n first channels arranged in a plane parallel to the device plane, wherein the microfluidic device further comprises a plurality of first side chambers, especially wherein each first channel is fluidly connected to one or more (individual) first side chambers. In further embodiments, especially at least one (individual)

first side chamber extends from any one of the first channels in a direction parallel to the plane.

Any one of the first side chambers may be accessible via a respective first opening via the first side or via the second side.

In further specific embodiments, each first side chamber is accessible via a respective first opening via the first side or each first side chamber is accessible via a respective first opening via the second side.

The plurality of first channels may be identical first channels.

If something is described herein in relation to one first channel (e.g. the configuration or the method to fabricate, etc.), this description may thus also relate to more than one first channel (especially to a plurality of (identical) first channels). With a system comprising a plurality of similar first channels, especially wherein two or more first side chambers extend from each first channel, wherein the side chambers comprise first electrode elements, multiple analytes or reactions or effects may be analyzed simultaneously using electrical measurement (like voltammetry or amperometry), in one experiment.

In further specific embodiments, the microfluidic device is a microfluidic cell culture device.

The system of the invention may in embodiments be used for measuring a barrier function.

In advantageous embodiments, the system, especially the microfluidic device further comprises one or more second channels, fluidly connected to the one or more of the first channels.

A barrier may be detined between the first channel and the second channel.

The barrier may e.g. be defined by biological cells (growing) at an interface between (or of) the first channel and the second channel.

The barrier (such as the cells) may be studied by determining an electrical signal (like current, resistance, capacitance, or any other relevant signal) over the barrier.

The second channel may be configured for hosting a second electrode element or for being fluidly connected or connectable to a second electrode element (allowing to measure an electrical signal over the barrier). In embodiments at least one (individual) (second) side chamber may extend from the second channel.

The second side chamber may be fluidly connected to the second side chamber(s). The second side chamber is also especially configured for hosting a second electrode element.

The second channel and the second side chamber(s) may in embodiments essentially be configured like the first channel and the first side chamber(s). In embodiments, the second channel (also) is fluidly connected to {one or more) (second) side chambers extending from the second channel.

In further embodiments, also the second side chamber(s) comprise a (second) electrode element.

As such, the barrier function may be studied by measuring an electrical signal between a first electrode element (in a first side chamber) and a second electrode element (in a second side chamber) (of respectively a first channel and a second that are fluidly connected to each other). The second side chamber is essentially not directly fluidly connected to the first side chamber(s). A fluid connection between any second side chamber and any first side chamber may especially only be provided via the second channel and the first channel.

Hence, in further embodiments, the microfluidic device further comprises (i) a second channel and a second side chamber extending from the second channel (especially at a location between a second channel inlet and a second channel outlet). Moreover, the microfluidic device especially further comprises (i) one or more second channels and one or more second side chamber.

Further, the second side chamber is (are) especially fluidly connected to (one of) the second channel(s). The second side chamber(s) especially extend(s) from (one of) the second channel(s) in a direction parallel to the device plane.

The (each) second channel is especially fluidly (and sealingly) connected to (one of) the first channel.

Further, in embodiments, the second side chamber is accessible via a second opening either via the first side or via the second side.

Especially each second side chamber is accessible via an (individual/respective) second opening either via the first side or via the second side.

Herein the terms “second channel” and “second side chamber” may relate to a plurality of (identical) second channels and a plurality of second side chamber, as is described above in relation to the first channel.

The term “second channel” may relate to n second channels.

The number n especially corresponds to the number n described in relation to the first channels.

Further, especially any individual first channel may be fluidly connected to an individual second channel (and vice versa). The plurality of second channels may comprise a plurality of comparable second channels (each one fluidly connected to one or more individual second side chambers). Further if anything is described herein in relation with one second channel, the same may relate to a plurality of second channels.

In further specific embodiments (comprising one or more first channels as described above), the microtluidic device further comprises (i) one or more second channels and one or more second side chamber.

Each second channel especially comprising a second channel inlet and especially also a second channel outlet.

Further, one or more of the second channel inlets may be fluidly connected to each other at a location upstream of the (respective) second fluid inlets.

Likewise one or more of the second channel outlets may be fluidly connected to each other at a location downstream of the second channel outlets.

The second channel inlets and the second channel outlets may be arranged comparable to the first channel inlets and outlets, see also above in relation to the first channels.

Further, especially each second channel bemg fluidly connected to one or more (individual) second side chambers extending from the (respective) second channel (especially at a location between the second channel inlet and the second channel outlet) (in a direction parallel to the device plane). Further, especially each second channel being fluidly (and sealingly) connected to a single first channel. Further, in embodiments, each second side chamber is accessible via an individual second opening either via the first side or via the second side.

The second opening (of any of the second side chambers) is further especially configured for receiving (at least part of) a second electrode element and configured for arranging at least part of the second electrode element in the second side chamber. In specific embodiments, the system further comprises one or more second electrode elements (each second electrode element) at least partly configured in (one of) the one or more second openings and (in) the (respective) second side chambers. The second opening is in embodiments configured as is described in relation to the first opening, see above. The second opening is especially configured for arranging the second electrode element in the second side chamber such that the second electrode element substantially does not disturb the environment in the second channel. The second electrode element (when configured in the second opening) is especially configured not to (physically and/or visually) block the second (and the first) channel. In embodiments, the second electrode element is configured substantially entirely remote from the second channel. In further embodiments, a first portion of the second electrode element configured in the second side chamber is larger than a second portion of the in the second electrode element configured in the second channel. The second electrode element may especially not extend into the second channel. Moreover, the second electrode element may also not extend in the first channel. Likewise, the first electrode element may not extend in the second channel.

Further, the configuration, shape and dimensions of the second side chamber(s) may correspond to the configuration, shape and dimensions described in relation with the first side chamber(s). As described above in relation to the first side chambers, in embodiments, the system may also comprise a plurality of second side chambers. Moreover, in specific further embodiments, the microfluidic device comprises two or more second side chambers. In further embodiments, each second side chamber is fluidly connected to the (respective) second channel. Further, especially each second side chamber extends from the second channel in a direction parallel to the device plane. Especially, each second side chamber is accessible via a respective second opening either via the first side or via the second side. Hence in embodiments, one or more (especially two or more) second side chambers extend from the second channel. The one or more second side chambers especially extend in a direction parallel to the device plane. Further, the one or more second side chambers are especially fluidly connected to the second channel. Especially, the one or more second side chambers are accessible via an individual second opening either via the first side or via the second side. The second channel may in embodiments have a shape and/or a dimension as described with respect to the first channel. The second channel may also be elongated and comprising a (longitudinal) second channel axis. The first channel and the second channel may in embodiments cross each other and at a junction (of the channels) the first channel may be fluidly connected to the second channel. This may e.g. be the case in a microfluidic device comprising a single first channel and a single second channel. In other embodiments, the first channel and the second channel may be arranged parallel to each other and over at least part of alength of the first channel and the second channel, the channels may be fluidly connected to each other. The second channel in combination with the first channel may define a double- channel with a (longitudinal) double-channel axis. Such double-channel may comprise a channel comprising a first compartment (defined by the first channel) and a second compartment (defined by the second channel). In further embodiments, the (longitudinal) first channel axis (of the first channel) is configured parallel to the (longitudinal) second channel axis (of the second channel). Especially, the first channel and the second channel together (optionally together with a membrane, see below) define a microfluidic double-channel (“double-channel”) with a double-channel axis. The double-channel axis may be parallel to the first channel axis and the second channel axis. The first channel axis and the second channel axis are especially not configured in single plane parallel to the first side or second side. In further embodiments (especially of the double channel) the first channel axis and the second channel axis are configured in a single plane perpendicular to the first side or second side. In alternative embodiments, the first channel axis and the second channel axis may be configured in single plane parallel to the first side or second side (see also below).

As described above in relation to the first channel, also the second channel not necessarily is circumferentially closed. Yet, the microfluidic double-channel is essentially circumferentially closed. At a first side, the first channel wall (at least partly surrounding the first channel) may enclose the double-channel. At another side, a second channel wall that at least partly surrounds the second channel may enclose the double-channel. The first channel may in embodiments be circumferentially closed by the second channel in combination with the first channel wall. The first channel may be closed at a first portion of the circumference by the first channel wall and at a second portion of the circumference (adjacent to the second channel) the first channel may be closed by the second channel wall or by e.g. a (second) fluid present in the second channel. Likewise, the second channel may in embodiments be partly closed by the first channel, such as by the first channel wall or by a (first) fluid in the first channel. The first fluid and the second fluid may e.g. be immiscible (e.g. based on a difference in surface tension, polarity and/or viscosity) and define an interface and the interface may actually be arranged at the location where the first channel contacts the second channel. In embodiments one of the first fluid and the second fluid comprises a hydrogel.

Yet, in further embodiments, a porous membrane is configured between the first channel and the second channel. Hence, the porous membrane may define the interface between the first channel and the second channel. The porous membrane may circumferentially close the first channel in combination with the first channel wall. Simultaneously, the porous membrane together with the second channel wall may circumferentially close the second channel. Further, the porous membrane may allow a fluid transport trough the membrane (to provide the fluid connection between the first channel and the second channel, and especially (also) to conduct electricity).

The porous membrane may in embodiments comprise (a plurality of) pores having a diameter equal to or smaller than 20 um, such as equal to or smaller than 15 um. The pore diameter is especially at least > 2pm. Further, the membrane may in embodiments have a thickness (perpendicular to a plane of the membrane) of at least 0.2 um, such as at least 0.5 um. The thickness of the membrane may especially be equal to or smaller than 1 mm, such as equal to or smaller than 0.5 mm, or even equal to or smaller than 100 um, e.g. in the range of

0.2-50 um, such as 0.5-10 um, especially 0.5-5 pm.

Hence, in further embodiments, the microfluidic device further comprises a porous membrane arranged for (physically) separating the second channel from the first channel, wherein the second channel is fluidly (and sealingly) connected to the first channel via the porous membrane. In embodiments, the porous membrane separates a plurality of first channels from a plurality of second channels. The plurality of second channels may especially be configured in a (single) plane parallel to the device plane.

The term “porous membrane” may also relate to a plurality of (different) porous membranes. In embodiments, e.g. a plurality of first channels and second channels are arranged alternatingly in the same plane and one porous membrane or a plurality of porous membranes are configured between each set of one first channel and one second channel (together defining one microfluidic double-channel). The porous membrane(s) may in embodiments be arranged perpendicular to the device plane (at the location of the microfluidic double-channel(s)). Especially, the porous membrane is arranged parallel to the device plane, especially wherein all first channels are configured in a first plane parallel to the device plane and all second channels are configured in a second plane parallel to the device plane.

In the depicted embodiments, the device may comprise a plurality of microfluidic double-channels.

Thus, the term “(microfluidic) double-channel” may especially also relate to a plurality of (microfluidic) double-channels.

In such embodiment, especially for each double-channel, the first channel axis and the second channel axis are configured in a respective single plane perpendicular to the first side or second side.

In further embodiments, the porous membrane is arranged parallel to de device plane.

Further, the plurality of microfluidic channels are especially (also) (all) arranged in a plane parallel to the device plane.

In further embodiments, the microfluidic device comprises n first channels, n second channels, m first side chambers, and p second side chambers, wherein m>n and wherein p >n, especially wherein n>2, wherein the first channels are (all) arranged in a plane parallel to the device plane, wherein at least one individual first side chamber extends from at least one of the first channels, especially any one of the first channels, in a direction parallel to the device plane.

Especially, each first channel is fluidly connected to one or more individual first side chambers.

Further, at least one individual second side chamber extends from at least one of the second channels, especially from any one of the second channels, in a direction parallel to the device plane.

Especially, each second channel is fluidly connected to one or more second side chambers.

Further, especially each first side chamber is accessible via an individual first opening either via the first side or via the second side and each second side chamber is accessible via an individual second opening either via the first side or via the second side.

In embodiments any one of the first channels is configured fluidly connected to a respective (individual) second channel, wherein said first channel and said second channel together define a respective microfluidic double-channel.

In further embodiments, any one of the first channels is configured fluidly connected to a respective(individual) second channel via the porous membrane (especially one or more porous membranes), wherein said first channel and said second channel (and the porous membrane (s)) together define a respective microfluidic double-channel.

The number n may in embodiments have a value as described above in relation to the number of first channels.

Further, especially (also) the second channels are (all) arranged in a second plane parallel to the device plane.

In further embodiments, the one or more first side chambers are (especially each first side chamber is) accessible (via an individual first opening) via a single side selected from the first side and the second side, and the one or more second side chambers are (especially each second side chamber is) accessible (via an individual second opening) via the (same) single side. In yet further embodiments, any one of the first channel inlets, the first channel outlets, the second channel inlets, and the second channel outlets is (also) configured at the (same) single side.

As described above, the device may be used for barrier measurements, Therefore, in embodiments, e.g. a resistance of the barrier (e.g. of cells at the interface between the first channel and the second channel) may be measured between a first side chamber and a second side chamber. The barrier may be formed by the cells, such as by a mono layer of cells, or, e.g., by tissue formed by the cells. Especially an individual first side chamber (fluidly connected to the first channel) and an individual second side chamber (fluidly connected to the second fluid channel) of at least one of the microfluidic double-channels (together) may define a two-chamber arrangement. Especially such two-chamber arrangement may be used for the measurement. In the two-chamber arrangement the first side chamber and the second side chamber may be aligned (at opposite sides of the double-channel axis). Hence a distance between the first side chamber and the respective second side chamber along the microfluidic double-axis may be zero. This may also be indicated as the first side chamber extends at a first longitudinal position from the microfluidic double-channel and the second side chamber extends from the microfluidic double-channel at the (same) first longitudinal position. Yet, the two chambers may in further embodiments be arranged at a distance from each other, and the distance between the first side chamber and the respective second side chamber along the microfluidic double-axis may be 10 mm or more, such as e.g. up to 20 mm. In further embodiments, in the two-chamber arrangement, a distance L12 between the first side chamber and the second side chamber along the microfluidic double-channel axis is selected from the range of 0-10 mm, especially 0-5 mm, such as 0-2 mm, even more especially 0-1 mm. In embodiments, the first side chamber extends at a first longitudinal position from the microfluidic double-channel and the second side chamber extends from the microfluidic double-channel at further longitudinal position of the microfluidic double-channel. In such embodiment, the distance between the first longitudinal position and the further longitudinal position may correspond to the distance L12.

Using a two chamber arrangement, e.g., a resistance over the barrier may be measured. The measured resistance may not only be the result of the barrier, but also of (the fluid in) the first channel and second channel and other e.g. systematically present elements. Therefore it may be advantageous to also measure a resistance in the first channel in isolation, and/or a resistance of the second channel in isolation and/or a resistance along another path between the first channel and the second channel (over the barrier). Hence, in further embodiments, measurements may be performed between a plurality of first side chambers and/or a plurality of second side chambers (of the same double-channel). Hence, one or more first side chambers and one or more second side chambers (of the same double-channel) together may define a 2-, 3-, 4-, 5-, 6-, etc.- chamber arrangement. In such chamber arrangement (successively) barrier measurement may be performed between two of the different chambers (especially electrode elements in the chambers).

For instance, a three-chamber arrangement may comprise a “V- configuration”, comprising e.g. two first side chambers at a first side of the double-axis and spaced apart from each other, together with one second side chamber arranged opposite to the two first side chambers and especially at the same distance from both the first side chambers. It will be understood that the (first and second) side chambers may be configured in any desirable configuration. The (first and second) side chambers especially may be configured to provide the respective electrode element at the desired location. In further embodiments, a four- chamber arrangement may comprise two first side chambers and two second side chamber, especially wherein one of the first side chambers is arranged opposite to one of the second side chambers, and wherein also the other first side chamber is arranged opposite to the other second side chamber. In further embodiments both first side chambers (as well as both second side chambers) are mutually arranged at opposite sides of the microfluidic axis. In other embodiments (of the four-chamber arrangement) both first side chambers are arranged at a first side of the double-channel and the second side chambers are arranged at the opposite side of the double-channel axis. Using (electrodes in) the four-chamber arrangement allows for 6 individual measurements that may be combined to give the final result. In embodiments, (i) two individual first side chambers extending from the microfluidic double-channel (especially from the first channel) and (ii) two individual second side chambers extending from microfluidic double-channel (especially from the second channel) of at least one of the microfluidic double-channels (together) define a four-chamber arrangement. In further embodiments, especially two of the side chambers extend at a first longitudinal position (from the microfluidic channel) and the other two extend at a second longitudinal position (from the microfluidic channel). The four-chamber arrangement may thus be configured in different ways. For instance, both first side chambers may extend at the first longitudinal position and both second side chambers may extend at the second longitudinal position. Or one of the first side chambers and one of the second side chambers extend at the first longitudinal position and the other first side chamber and the other second side chamber extend from the second longitudinal position. In further specific embodiments, (i) two individual first side chambers extending in opposite directions from the microfluidic double-channel (especially from the first channel) and (ii) two individual second side chambers extending in opposite directions from microfluidic double-channel (especially from the second channel) of at least one of the microfluidic double-channels (together) define a four-chamber arrangement. In further embodiments (of the four-chamber arrangement) one of the first side chambers and one of the second side chambers extend (from the microfluidic double-channel) at the first longitudinal position (in opposite directions), and another one of the first side chambers and another one of the second side chambers extend (from the microfluidic double-channel) at the second longitudinal position (in opposite directions). Alternatively, (both of) the first side chambers extend (from the microfluidic double-channel) at the first longitudinal position (in opposite directions), and (both of) the second side chambers extend (from the microfluidic double- channel) at a second longitudinal position (in opposite directions).

In further embodiments, (in the four-chamber arrangement) a distance L11 between the first longitudinal position and the second longitudinal position is (selected from the range of) larger than zero, and especially smaller than 20 mm, such as smaller than 10 mm. The distance L11 is especially in the range of 0.05-4 mm, such as 0.1-2 mm.

It may further be advantageous to configure more than one chamber arrangement at different locations along the microfluidic axis. In further embodiments, the microfluidic device comprises a plurality of two-chamber arrangements and/or a plurality of four-chamber arrangements distributed (adjacently) along the microfluidic double-channel axis (of one or more of — especially any one of- the microfluidic double-channels).

Hence, in embodiments, the system comprises one or more first electrode elements and one or more second electrode elements. Further, in embodiments in at least one microfluidic double-channel (i) in one or more first side chambers the first electrode element is at least partly configured in the first opening and the first side chamber, especially wherein the first electrode element does not extend into the first channel, and (ii) in one or more second side chambers the second electrode element is at least partly configured in the second opening and the second side chamber, especially wherein the second electrode element does not extend into the second channel.

In further embodiments, in at least one two-chamber arrangement and/or in at least one four-chamber arrangement (i) in any one of the first side chambers one first electrode element is at least partly configured in the first opening and the first side chamber (especially wherein the first electrode element does not extend into the first channel), and (ii) in any one of the second side chambers the (especially one) second electrode element is at least partly configured in the second opening and the second side chamber (especially wherein the second electrode element does not extend into the second channel). Further, for allowing a visual inspection of the double-channel, in embodiments (also) the second channel wall is transparent.

The second channel wall may be defined by a second layer (optionally in combination with a further later), see below.

Hence, especially the second layer is transparent.

The second layer (and/or second channel wall) may comprise a transparent material, such as described herein in relation to the first layer.

The second layer may (also) comprises a glass layer.

The second layer (and/or second channel wall) may in further embodiments (further) comprise a second transparent polymeric material.

The second polymeric material may differ from the first polymeric material.

Especially, (also) the porous membrane is transparent.

Also, the porous membrane may comprise a transparent material as described in relation to the first layer.

In further specific embodiments, the microfluidic device comprises (1) a first layer comprising the one or more first channels and the one or more first side chambers, and (ii) a second layer comprising the one or more second channels and the one or more second side chambers, wherein the first layer, the second layer and the optional membrane are at least partly transparent (for visible light). In embodiments, a line of sight is provided from external of the microfluidic device to at least part of one or more of (i) the first channel, (ii) the one or more first side chambers, (iii) the second channel, (iv) the one or more of the second side chambers.

In further embodiments, the microfluidic device comprises a first layer comprising the one or more first channels and the one or more first side chambers, and the first layer is at least partly transparent (for visible light). In embodiments, a line of sight is provided trom external of the microfluidic device at least part of one or more of the first channel and the one or more of the second side chambers.

The invention further provides the method for fabricating a system comprising a microfluidic device, especially wherein the method comprises a preparation stage and a connecting stage.

In specific embodiments the connecting stage comprises (i) providing a first layer with a primary first layer face comprising a continuous (elongated) first recess, wherein the first recess comprises a first side extension (extending away from the first recess) (especially along the primary first layer face) and (ii) covering the first recess with a cover, wherein a first cover side closes the continuous first recess, especially to provide the microfluidic device comprising a first channel and a first side chamber extending from the first channel.

Further, in specific embodiments, the preparation stage comprises (i) providing a first opening from a secondary first layer face (arranged opposite to the primary first layer face) to (the primary first layer face at a location of) the first side extension or (ii) providing the first opening from the first cover side to a second cover side (especially opposite to the first cover side) at a location of the cover, especially of the first cover side, configured for covering the first side extension.

Especially, the preparation stage is configured before, during, and/or after the connecting stage.

In embodiments, the secondary first layer face may define the first side of the microfluidic device.

In further embodiments the cover may define the second side of the microfluidic device.

The term “covering” in the phrase “covering the recess with the cover” and the like especially relates to closing the recess, especially wherein the cover seals the recess.

The first opening may in specific embodiments be provided before applying the cover.

The first layer may comprise a (thermoplastic) polymeric material, for instance as described above.

In a thermoplastic polymeric material, the opening may e.g. be provided by puncturing or piercing a hole in the first layer.

In further embodiments, the first layer comprises glass or a hard plastic, such as polycarbonate.

In such material the first opening may tooled or drilled.

To prevent any material that may come loose when providing the opening in the layer, the preparation stage may be configured before and/or optionally during the connecting stage.

Further, in embodiments, in the preparation stage also a first channel inlet and a first channel outlet may be provided.

In embodiments the preparation stage further comprises providing a first channel inlet and a first channel outlet through the first layer from the secondary first layer face, into the continuous first recess, especially at locations of extremes of the first recess or (ii) providing the first channel inlet and the first channel outlet through the cover from the first cover side to the second cover side at locations of the cover configured for covering the extremes of the first recess.

If the first recess is a continuous recess, the cover may be a continuous cover.

Yet, in embodiment, the method may provide the microfluidic device comprising a plurality of first channels, see below.

For such embodiments, the cover may also comprise a number of (discrete) cover sections (together defining the cover). Hence, the term “cover” may refer to a plurality of covers.

The cover may in embodiments comprise a (second) layer.

The second layer may also comprise a (continuous) recess to provide a second channel to the microfluidic device.

In further embodiments, the cover comprises a second layer comprising a primary second layer face comprising a (continuous) (elongated) second recess. Especially, the second recess comprises a second side extension {extending away from the second recess) (along the primary second layer face). In embodiments, the primary second layer face is connected to the primary first face in the connecting stage. Hence, in embodiments, the first cover side comprises the primary second layer face. In other embodiments, a porous membrane is arranged between the primary second layer face and the primary first face when covering the first recess in the connecting stage. Hence, in embodiments, the first cover side comprises the primary second layer face. In other embodiments, the cover further comprises a porous membrane arranged at the primary second layer face, and the first cover side comprises the porous membrane. Hence, in embodiments the cover comprises the second layer and porous membrane arranged over the primary second layer face. Further, especially the connecting stage further comprises fluidly connecting the first recess to the second recess (optionally via the porous membrane), wherein the first layer closes the (continuous) second recess thereby defining a second channel and a second side chamber extending from the second channel, especially wherein the second side chamber is fluidly connected to the second channel. The second recess may especially define the second channel and the second side chamber(s).

Further, the second recess especially comprises the second channel axis. The preparation stage may further especially comprise (1) providing a second opening (through the first layer) from the secondary first layer face to the primary first layer face, at a location configured for being covered by the second side extension of the cover and especially further (when the porous membrane is applied) providing the second opening through the optional membrane, such that the second opening in the first layer and in the porous membrane are aligned in the (provided) microfluidic device or (ii) providing the second opening (through the cover) from the secondary second layer face to (the primary second layer face at a location of) the second side extension. The second opening may be provided before, during and/or after the connecting stage.

Further, in embodiments, in the preparation stage also a second channel inlet and a second channel outlet are provided. In embodiments the preparation stage further comprises providing a second channel inlet and a second channel outlet through the first layer from the secondary first layer face, at locations configured for being covered by extremes of the second recess of the cover and further (when the porous membrane is applied) providing the second channel inlet and second channel outlet through the optional membrane, such that the second channel inlet and the second channel outlet in the first layer and in the porous membrane are aligned in the provided the microfluidic device or (ii) providing the second channel inlet and the second channel outlet through the cover from the secondary second layer face into the (continuous) second recess at locations of extremes of the second recess. The method may provide the microfluidic device comprising one or more microfluidic double-channels. Hence, in further embodiments, the first recess comprises a (longitudinal) first recess axis and the second recess comprises a second (longitudinal) recess axis. Especially the first recess axis is arranged parallel to the second recess axis when fluidly connecting the first recess to the second recess in the connecting stage, thereby providing a microfluidic double-channel comprising a (longitudinal) double-axis.

In further advantageous embodiments the primary first layer face comprises n first recesses wherein each first recess comprises at least one first side extension (extending away from the respective recess), and the primary second layer face comprises n second recesses, wherein each second recess comprises at least one second side extension (extending away from the respective recess). For such embodiment, the connecting stage especially comprises fluidly connecting each of the first recesses to an individual second recess wherein the first cover side closes each of the first recesses and the first layer closes each of the second recesses, thereby defining (i) n first channels, wherein each first channel comprises at least one first side chamber extending from the first channel (and especially being fluidly connected to the first channel) and (ii) n second channels, wherein each second channel comprises at least one second side chamber extending from the second channel and being fluidly connected to the second channel. Further, the preparation stage especially comprises (i) providing a respective first opening (through the first layer) from the secondary first layer face to (the primary first layer face at the location of) each of the first side extensions and providing a respective second opening (through the first layer) from the secondary first layer face to the primary first layer face at locations configured for being covered by the second side extension of the cover and further optionally (when the optional membrane is applied) providing the second openings through the optional membrane, such that the second openings in the first layer and in the porous membrane are aligned in the (provided) microfluidic device, or (ii) providing the first openings (through the cover) from the first cover side to the second cover side at each of the locations of the cover configured for covering the first side extensions and providing the second openings (through the cover) from the secondary second layer face to (the primary second layer face at the locations of) each of the second side extension. Especially, the first openings and the second openings are provided before, during and/or after the connecting stage.

Further, especially, any first channel inlet and any first channel outlet is provided to each of the first channels and any second channel inlet and any second channel outlet is provided to each of the second channels.

In further embodiments, wherein the first side (of the device) is configured closest to the secondary first layer face and the second side (of the device) is arranged closest to the secondary second layer face, in the preparation stage the first opening in any first side chamber is configured for accessing the first opening via a single side selected from the first side and the second side, and the second opening in each second side chamber is configured for accessing the second opening via the single side.

In further specific embodiments, the method further comprises (i) providing a first electrode element at least partly in at least one of the first openings and the respective first side chamber, especially wherein the first electrode element does not extend into the first channel; and (ii) providing a second electrode element at least partly in at least one of the second openings and the respective second side chamber, especially wherein the second electrode element does not extend into the second channel.

The method may further comprise one or more stages for providing any one of the recesses is the first and/or second layer. The first and/or second layer may comprise a hard plastic, such as polycarbonate. In such material recesses and/or openings and/or channel inlets or outlets may especially be milled. Alternatively or additionally, thermoplastic materials may be selected that may be casted, Hence, in embodiments, the method may further comprise (i) casting a first polymeric material in a first mold configured for providing the first layer and curing the first polymeric material in the first mold to provide the first layer, and (ii) casting a second polymeric material in a second mold configured for providing the cover and curing the second polymeric material in the second mold to provide the cover. During casting especially, therecess(es) for the channel(s) and the side chamber(s) may be configured. Feasible polymeric materials, for the first layer and the second layer are described herein. The first polymeric material may e.g. comprise PDMS (polydimethylsiloxane). Additionally, or alternatively the second polymeric material comprises PDMS. In embodiments, (also) the porous membrane comprises a polymeric membrane material comprises PDMS.

In yet a further aspect, the invention provides a method for analyzing one or more of a biological cell and biological tissue. The method especially comprises providing a system as described herein, or obtainable by the method described herein for fabricating a system. In embodiments, the method further comprises providing the biological cell (in a fluid) to at least one first channel, wherein at least one first side chamber comprising or functionally coupled to a first electrode element extends from (and is fluidly connected to) the at least one channel. Further, especially a further electrode element is arranged or functionally (fluidly and/or conductively) coupled to the first channel or to the second channel. The further electrode may e.g. be arranged inside the (first or second) channel or may be downstream of the channel. Additionally or alternatively, the method may further comprise providing the biological cell (in a fluid) to at least one second channel fluidly connected to a second side chamber comprising or functionally coupled to a second electrode element. Herein the term a “(biological) cell” may refer to a plurality of (different) (biological) cells. In embodiments the term may refer to a “monolayer of cells” or a “cell monolayer”. In further embodiments, the term may refer to a biological tissue comprising biological cells. The monolayer and/or the tissue may grow from one or more biological cell (e.g. after being provided to the first and/or second channel).

The method may therefore also be indicated as “a method for analyzing one or more of a biological cell, a cell monolayer, and biological tissue”.

In further embodiments, the method further comprises imposing an electrical signal to (the fluid in) the at least one further electrode and measuring a transformed electrical signal at the first electrode element (or at the second electrode element) and/or imposing an electrical signal to the first electrode element (or second electrode element) and measuring a transformed electrical signal at the further electrode element. The method may further comprise determining the property of the biological cell (including a plurality of biological cells) based on the electrical signal and the transformed electrical signal. The biological cell may further form a biological tissue. The biological cell may form a (cell) monolayer. The biological cell, especially the plurality of biological cells may comprise (or define) the (cell) monolayer. Hence, the method may further comprise determining the property of the biological tissue based on the electrical signal and the transformed electrical signal. Especially, the method may further comprise determining the property of one or more of the biological cell{s) (including the cell monolayer) and the biological tissue based on the electrical signal and the transformed electrical signal.

In further specific embodiments, the method comprises providing the system, wherein the microfluidic device comprises at least one microfluidic double-channel, especially wherein a first electrode element is configured in at least one first side chamber (extending from the microfluidic double-channel) and a second electrode element configured in at least one second side chamber (extending from the microfluidic double-channel); growing biological tissue in one or more of the first channel and the second channel of the at least one microfluidic double-channel; imposing the electrical signal to the first electrode element and /or to the second electrode element, and measuring a transformed electrical signal at the second electrode element and/or the first electrode element to provide a transepithelial electrical resistance (TEER) measurement of the tissue over time.

In further specific embodiments the system comprises a microfluidic device comprising a plurality of microfluidic double-channels, wherein each of the microfluidic double-channels comprises a four-chamber arrangement, wherein in all first side chambers a respective first electrode element is at least partly configured in the first opening and the first side chamber, and wherein in all second side chambers a respective second electrode element is at least partly configured in the second opening and the second side chamber, and wherein the method further comprises performing a TEER measurement in each of the microfluidic double-channels, wherein repeatedly an impedance is measured between two of the electrode elements of the four-chamber arrangement.

Herein the terms “imposing an electrical signal” and measuring a “transformed signal” may especially relate to any electrical measurement. The signal may be imposed to both of the electrodes and also both electrodes may be used to measure the transformed signal. The signal may further change over time. For instance, a resistance may be measured by providing a direct current to one of the electrodes. Yet, also the impedance between the electrodes may be measured, as such also an alternating current may be provided to the first and the further electrode. The electrical measurement may comprise voltammetry or amperometry.

The terms “upstream” and “downstream” relate to an arrangement of items or features relative to the propagation of an element such as a particle or a fluid in a channel or light in a beam of light (during operation), wherein relative to a first position within the channel or beam, a second position in the channel or beam closer to an inlet (for the element or fluid) of the channel or respectively closer to a light generating means is “upstream”, and a third position within the channel further away from the inlet or respectively further away from the light generating means “downstream”.

The term “controlling” and similar terms herein especially refer at least to determining the behavior or supervising the running of an element, especially wherein the element is configured to adjust the treating of the damages skin tissue. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the (controllable) element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc., especially actuating. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element. The controlling of the element can be done with the control system. The control system and the (controllable) element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise at least part of the control system. In embodiments, the control system and element may not be physically coupled. Control can be done via wired and/or wireless control. The term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one control system may be a master control system and one or more others may be slave control systems.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1 schematically depicts aspects of a system of the invention; Figs 2A-2B schematically depict some further aspects of the system; Fig. 3A-3B schematically depict aspects of the microfluidic device; and Figs 4A and 4B schematically depict some further aspects of the invention. The schematic drawings are not necessarily to scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS Fig. 1 schematically depicts an embodiment of the system 1000 comprising a microfluidic device 1. The microfluidic device I comprises a first channel 100 having a first channel inlet 101 and a first channel outlet 102 and two first side chambers 150 extending from the first channel 100 in a direction of the device plane 6. The first side chambers 150 are fluidly connected to the first channel 100. The microfluidic device 1 further comprises a first side 2 and a second side 3 with the device plane 6 in between. The device plane 6 is a virtual plane as is indicated by the dotted lines. The plane 6 may especially be parallel to the first side 2 and/or the second side 3, see e.g. Fig 3A. In the embodiment, both first side chambers 150 are accessible via the (individual) first opening 151 via the first side 2. In other embodiments one or more of the first side chambers 150 may be accessible from the second side 3. Here, the first channel inlet 101 and outlet 102 are also accessible via the second side 2. In further embodiments, they may extend in a direction of the first fluid axis 105.

In Figs 2A-2B, an embodiment of the system 1000 is depicted, wherein the microfluidic device 1 further comprises a second channel 200. Moreover, the depicted embodiment comprises three first channels 100 and three second channels 200. Further, from each second channel 200 two second side chamber 250 extend. The second side chambers 250 are fluidly connected to the second channel 200 and extend from the second channel 200 in a direction parallel to the device plane 6. As further can be seen, the second side chambers 250 are fluidly connected to the second channel 200. As is clearly depicted in Fig. 2B, the second channel 200 is fluidly connected to the first channel 100. Further, the second side chamber 250 is accessible via a second opening 251 via the first side 2 (in other embodiments the second side chamber may be accessible via the second side 3). Fig. 2B further depicts that the system 1000 further comprises one or more first electrode elements 8 configured in the first opening 151 and the first side chamber 150 and a second electrode element 9 is configured in the second opening 251. In Fig 2B, the first electrode element 8 does not extend into the first channel 100 or in the second channel 200. Likewise, the second electrode element 9 does not extend in the second channel or the first channel 100. This way, the electrode elements 8, 9 may not interfere with the process in the channels 100, 200. Further, when the elements of the microfluidic device 1 are transparent, the channels 100, 200, and e.g. biological cells 2001 in the channels 100, 200 may visually be inspected. The embodiment is an example of a system 1000, especially a device 1, wherein the one or more first side chambers 150 are accessible via a single side selected from the first side 2 and the second side 3, and wherein the one or more second side chambers 250 are accessible via the single side. In the embodiment, both side chambers 150 and 250 are accessible via the first side 2. The device 1 depicted in Fig. 2B further comprises a porous membrane 300 that physically separates the second channel 200 from the first channel 100. The membrane 300 is arranged parallel to the device plane 6. The second channel 200 is fluidly connected to the first channel 100 via the porous membrane 300. The porous membrane 300 has a plurality of pores 310 having a diameter 311. In the figure, the size 311 of the pores 310 is exaggerated. The pores may especially be equal to or smaller than 20 pm. Further, the membrane 300 may in embodiments have a thickness 320 of at least 0.2 um. It is further schematically indicated that at one side of the membrane 300, in the first channel 100, a plurality of biological cells 2001 have formed (biological) tissue 2000. The depicted tissue 2000 shows a three-dimensional shape and may e.g. represent gut tissue 2000 formed by Caucasian colon adenocarcinoma (Caco2) cells 2001. In further embodiments the cells 2001 may form a (mono)layer of cells

2001. Hence the term “(biological) tissue” may in embodiments relate to a cell layer. Further,

the term biological cell 2001 may relate to a plurality of biological cells 2001, and e.g. to a monolayer of cells 2001/ a cell monolayer.

The embodiments of Figs 2 further depict a microfluidic double-channel 10 with a double-channel axis 15, In the embodiments, the first channel axis 105 is configured parallel to the second channel axis 205 (and to the double-channel axis 15).

The device 1 depicted in Fig. 2A comprises three first channels 100, three second channels 200, six first side chambers 150, and six second side chambers 250. Hence, the number n of first channels 100 {and of second channels 200) >2; the number m of first side chambers 150 is equal to or larger than n (m2n) and also the number p of second side chamber 250 is equal to or larger than n (p>n). In the embodiment, the first channels 100 are arranged in a first plane parallel to the device plane 6 and the second channels 200 are arranged in a plane parallel to the device plane 6. Further from each first channel 100 two individual first side chamber 150 extend (in a direction parallel to the device plane 6). Likewise, from each second channel 200 two individual second side chamber 250 extend in a direction parallel to the device plane 6. Furthermore, (see insert of Fig. 2A) each first side chamber 150 is accessible via an individual first opening 151 via the first side 2 and each second side chamber 250 is accessible via an individual second opening 251 via the first side 2 (assuming that this is a view at the first side 2). In the embodiment, further any one of the first channels 100 is configured fluidly connected to a respective second channel 200, wherein the three first channels 100 and the three second channels 200 together define three microfluidic double-channels 10.

Further, Fig. 2B. see also Figs 3A-3B, also shows that the microfluidic device 1 comprises a first layer 19 comprising the one or more first channels 100 and the one or more first side chambers 150, and a second layer 29 comprising the one or more second channels 200 and the one or more second side chambers 250.

In Fig. 2A further some (embodiments of) chamber arrangements are indicated. In each of the microfluidic double-channels 10 an individual first side chamber 150 fluidly connected to the first channel 100 and an individual second side chamber 250 fluidly connected to the second fluid channel 200 together define a two-chamber arrangement 25. Actually, in the depicted embodiment each of the microfluidic double-channels 10 comprises four two- chamber arrangements 25. In the double-channel 10 indicated at the top, the two two-chamber arrangements 25 of side chambers 150, 250 arranged at opposite sides of the double-channel axis 15 are indicated with reference 25. In the insert one of the two further two-chamber arrangement 25 of side chambers 150, 250 arranged at two different longitudinal positions of the double-channel 10 is indicated with reference 25. Further also the distance L12 between the first side chamber 150 and the second side chamber 250 of the two-chamber arrangement 25 along the microfluidic double-channel axis 15 is indicated. The distance L12 relates to the distance between the first side chamber 150 and the second side chamber 250 (of the two- chamber arrangement 25), and especially to the distance between (a center of) the first opening 151 of the first side chamber 150 and (a center of) the second opening 251 of the second side chamber 250. In the depicted embodiments this distance L12 is 0 mm for the arrangement 25 of the oppositely arranged first and second side chambers 150, 250. Yet, for the adjacently arranged first side chamber 150 and second side chamber 250 as depicted in the insert, this distance L12 is non-zero.

The figure may also be used to explain an embodiment of the four-chamber arrangement 45. In such arrangement 45 two individual first side chambers 150 extending from the double-channel 10 (especially first channel 100) and two individual second side chambers 250 extending from the double-channel 10 (especially second channel 200) define the four- chamber arrangement 45. In the depicted embodiment one first side chamber 150 and one second side chamber 250 are (both) configured at a first longitudinal position 21 of the microfluidic double-channel 10, and the other first side chamber 150 and the other second side chamber 250 are (both) configured at a second longitudinal position 22 of the microfluidic double-channel 10. In the insert of Fig. 2A further the distance Lil between the first longitudinal position 21 and the second longitudinal position 22 (of the double-channel 10) is indicated. The depicted embodiment of the four-chamber arrangement 45 basically comprises two two-chamber arrangements 25 (arranged at opposite sides of the double-channel 10). Therefore, in depicted figure L12 equals L11 in size. However, both distances L11, L12 refer to different distances. L11 relates to the distance between the first longitudinal location 21 and the second longitudinal location 22. L12 relates to the distance between the first side chamber 150 and the second side chamber 250. In the embodiment, the first opening 151 and the second opening 251 are arranged parallel to the first longitudinal position 21 and the second parallel position 22, respectively. This may explain why L11 and L12 are of the same length in the embodiment.

Figs 3A-3B depicts some embodiments of the microfluidic device 1 in exploded view. The figures may also further explain the method of fabrication. The method may comprise a preparation stage and a connecting stage. In the connecting stage a first layer 19 with a primary first layer face 191 is provided. The first layer 19 comprises a continuous elongated first recess 192 with at least one first side extension 154. The first recess 192 is covered with a cover 290 to provide the microfluidic device 1, wherein a first cover side 297 closes the continuous first recess 192. When closing the first recess 192, the first channel 100 and a first side chamber 150 extending from the first channel 100 are defined. In the depicted embodiments, the cover 290 comprises a second layer 29 comprising a primary second layer face 291 with a second recess 292. The second recess 292 comprises a second side extension

254. As such, the first cover side 297 may comprises the primary second layer face 291, see Fig. 3A. In other embodiments as depicted in Fig. 3B, the cover 290 further comprises a porous membrane 300 arranged at the primary second layer face 291, and the first cover side 297 comprises the porous membrane 300. For these embodiments, the connecting stage further comprises fluidly connecting the first recess 192 to the second recess 292, optionally via the porous membrane

300. By connecting the recesses 192, 292, the first layer 19 closes the second recess 292 and the second channel 200 with one or more the second side chambers 250 extending from the second channel 200 are defined. The preparation stage for the given embodiment of Fig. 3B may have comprised: providing the first openings 151 through the first layer 19 from the secondary first layer face 196 (opposite to the primary first face 191), into the continuous first recess 192 at a location of the first side extension 154. Yet the opening may also have been provided from the other side (the primary first face 191). The first openings 151 may have been provided from first side extension 154 through the first layer 19 to the secondary first layer face 196. The first openings may e.g. have been punched or drilled from one first layer face to the other first layer face. Even by starting from each of the layer faces the first layer face openings 151 could have been provided. The term “providing the openings” in relation to from a first location to a second location merely indicates that the result of this action is an opening from the first location to the second location {or vice versa). The term does not indicate a direction in which the opening is provided (such as punched or drilled). Further, second openings 251 are provided through the first layer 19 from the secondary first layer face 196 at a location configured for being covered by the second side extensions 254 of the cover 290 and further the second openings 251 are further provided through the optional membrane 300, such that the second opening 251 in the first layer 19 and in the porous membrane 300 are aligned in the microfluidic device 1. In the preparation stage for the given embodiment also the first channel inlets 101 and the first channel outlets 102 are provided in the first layer 19. And the second channel inlets 201 and the second channel outlets 202 are provided through the first layer 19 and the porous membrane

300. The preparation stage for the depicted embodiment, is provided prior to the connecting stage.

In Fig. 3A further, the first recess axis 195 of the first recess 192 and the second recess axis 295 of the second recess 292 are indicated. After connecting the recesses 192, 292, they together provide the microfluidic double-channel 10 with the double-channel axis 15. It is further indicated that the first channel inlet 101, the first channel outlet 102, the second channel inlet 201, and the second channel outlet 202 not necessarily are configured at the first side 2 or the second side 3. They may also be configured at the edge of the microfluidic device Figs 4A-4B schematically depict a first mold 18 for providing the first layer 19 and a second mold 28 for providing the cover 290, in the depicted embodiment a second layer

29. In the first mold 18 a first polymeric material 11 may be casted and cured for providing the first layer 19 with three first recesses 192. In the second mold 28 a second polymeric material 21 may be casted and cured for providing the second layer 29 also comprising three second recesses 292. These molds 18, 28 may e.g. be used for providing the first layer 19 and second layer 29 depicted in Fig. 3B.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” includes also embodiments wherein the term “comprises” means “consists of”.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of" but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species”.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. In embodiments, the system comprises the control system. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

EXPERIMENTAL Experimentally embodiments of the system have been fabricated and TEER measurements have been performed as described below. Material and methods Chip fabrication A schematic illustration of the chip comprising an organ-on-chip device (“00C”) is shown in Figs 2. Two Poly(methyl methacrylate) (PMMA) molds for top and bottom channels were fabricated using micromilling (Datron Neo, Germany). PDMS base and curing agent were mixed (10:1 %w/w, Sylgard 184 Silicone elastomer kit, Dow Corning) and degassed. The PDMS was cast on the two PMMA molds and cured for 4 h at 60°C. In- and outlets were punched in the PDMS top layer with a 1 mm biopsy puncher. The inlets for the platinum electrodes (E1-E4; see Fig. 2A wherein the inlet for the respective electrodes are indicated with E1'-E4') were punched with a 0.5 mm puncher. After punching, any potential dust at the surface can be removed using Scotch tape. A PDMS membrane with a thickness of 2 um, was fabricated: an array of columns was formed with positive photoresist (PR) (AZ 9260) using a standard soft-lithography technique. Next, a solution of PDMS: Hexane (2:5 %w/w) was spin-coated on top of the wafer with PR columns and cured for 4 h. Finally, the PR was removed with acetone, releasing the membrane which was bonded by oxygen plasma treatment (Femto Science, Cute) to the PDMS top layer. The PDMS membrane was removed from the inlets of the bottom channel, and the electrode holes for the bottom channel (El and E4) to provide proper access. Next, the bottom layer was bonded to the membrane of the top layer using oxygen plasma. Four platinum wires (0.25 mm diameter, Alfa Aesar, Thermo Fisher Scientific) were cleaned and inserted in the pre-punched holes and secured using a UV curing glue (NOA). To completely cure the NOA, the chips were baked for 4h at 60°C. Cell culture Caucasian colon adenocarcinoma (Caco2) cells were used in the presented gut- on-chip. Caco2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose Glutamax medium (Gibco), supplemented with 20% fetal bovine serum (FBS, Gibco), and 100 U/L penicillin, 100 pg/mL streptomycin. The cells were cultured in T25 or T75 culture flasks and incubated at 37°C in humidified air (5% COz). Before cell seeding, the PDMS chips were treated with oxygen plasma (40 seconds, 50 Watt, Femto Science, Cute), flushed with 70% ethanol, and subsequently flushed with phosphate-buffered saline (PBS, Sigma-Aldrich). The channels were coated with 10 pg/mL collagen-1 (Gibco) in PBS for 30 minutes at 37°C. The collagen solution was replaced by cell culture medium and the chips were incubated for an additional 2 hours at 37°C and 5% CO:2.

Caco-2 cells were obtained from a T75 flask grown until nearly confluency, using 1x trypsin (Gibco) and resuspended in the cell culture medium. Caco-2 cells were seeded in the culture channels with a cell density of 5-10* cells/em?. After seeding, the chips were incubated for 1 hour at 37°C, subsequently, the cell medium was replaced by placing empty 200 pL pipette tips in the outlet and medium filled 200 uL pipette tips in the inlets. The medium was refreshed daily, after impedance measurements, by gravity-driven flow. The culture was maintained for 21 days (at 37°C, 5% CO) and cells were monitored by using phase-contrast microscopy (EVOS, FL Cell Imaging System, Life Technologies and LEICA DM IRM HC, air objectives). For the blank chips, the exact same procedure was followed, without the cell seeding step described in the previous paragraph. The blank chips were incubated at 37°C, 5% CO: for 7 days.

EGTA treatment On day 19, the cells were treated with a solution of 5 mM ethylene glycol-bis(B- aminoethylether)-N,N,N’ N’-tetraacetic acid (EGTA) in PBS for 45 min. Subsequently, the cells were incubated in cell culture medium to study the recovery of the barrier function. The impedance measurements were performed after 30 minutes and 1 hour of incubation. Impedance spectroscopy Prior to measuring, the cell culture medium in every compartment of the chip was replaced with DMEM at room temperature (RT). Impedance spectra were recorded every day using the Zurich Instruments HF2IS Impedance Spectroscope (Zurich, Switzerland). The impedance was recorded using an alternating current (AC) with an amplitude 0.1 V for six combinations of the four electrodes indicated in Fig. 2A (electrode combinations: E1-E2, El- E3, E1-E4, E2-E3, E2-E4, E3-E4) at a frequency range 100 Hz — 1 MHz. After recording the impedance spectra, the cell culture medium was replaced with DMEM at 37°C and the chips were placed in an incubator (37°C, 5% CO»).

The measured data was processed in MATLAB and TEER was obtained by determining a suitable readout frequency from impedance and phase plots. The information of the cell barrier was found from the four measurements between top-bottom electrode pairs,

which measure trough the membrane and cell layer (electrode pairs E1-E2, E1-E3, E2-E4, E3- E4). The magnitudes of these four electrode pairs are averaged per day and hereinafter referred to as [Zavl.

Correction factor The measured magnitude between the bottom-bottom (E1-E4) electrode pairs represents only the resistance of the medium (Rmea), as there are no cells cultured in the bottom channel, and this magnitude should theoretically always be the same.

The changes we do see in these bottom-bottom measurements are an indication to changes in Rmed, temperature or conductivity changes of the medium.

As the bottom and top channels have different dimensions, the medium resistance between the bottom-bottom (E1-E4) electrode pair differs from the medium resistance between a top-bottom (e.g.

E1-E2) electrode pair.

Therefore, the absolute change in medium resistivity cannot be used to correct for the previously mentioned factors that influence the Rmea and thus the impedance.

We can use the relative change in the measured bottom-bottom resistance, hereinafter referred to as the ‘correction factor’. Correction factor = ze, (1) bottom-bottOMgayo With this correction factor, we can correct the average magnitudes of the four top-bottom ([Zav|) electrode pairs via the following formula: |Zaraays| \Z correcteal = A — |Zav aay] (2) A lower temperature results in a higher medium resistance in the whole chip, and thus also to a higher magnitude measured between the bottom-bottom electrode pair (Zootom-bettom), which results in a correction factor > 1 using Formula 1. The corrected magnitude of the electrode pairs through the membrane will become lower by dividing with this correction factor (Formula 2). Results and discussion Fabricated chip We designed a microfluidic organ-on-chip device that consists of two PDMS parts separated with a porous PDMS membrane and contains three parallel cell-culture chambers (according to Fig. 1). Each cell-culture chamber was 1 mm wide with a I mm high and 24 mm long top chamber and a 0.2 mm high and 30 mm long bottom chamber.

The complete device was assembled by only using a plasma activation of the PDMS parts and did not require additional glues for bonding the membrane.

The 4 electrodes were inserted on the sides of the assembled chip and fixed with the NOA glue. The glue filled in the punched holes was immediately cured with the UV-lamp to prevent from its coverage of the electrodes and leakage to the culture chamber. This position of the electrodes allows inspection of the cell culture inside the culture channels and the electrodes can be easily multiplexed. By gluing the electrodes in the device, measurement errors due to variations in electrode placement are prevented.

Impedance measurements on-chip Prior to cell seeding, the chips were coated with a collagen-I and the impedance spectra were recorded in the blank chips filled with RT DMEM. Next, the Caco2 cells were seeded in the top compartment and were let to attach for 1 h. Subsequently, the culture medium was refreshed with RT DMEM and the impedance spectra were recorded. After recording the impedance spectra, the cell culture medium was replaced with 37°C DMEM and the chips were placed in the incubator. The process of the insertion of RT DMEM, recording impedance spectra, replacement with 37°C DMEM and subsequent incubation was performed for 21 days on every day.

Experimentally it appeared that for this setup and cell type, at approximately 2 kHz the maximum difference in the impedance spectra of empty chip compared to a chip with confluent cell layer was seen and therefore this was chosen as readout frequency. This frequency also corresponds with the crossing point of the phase plot for a blank chip and chip with cells; a method of determining the TEER known in the art. Concluding, during cell culture the impedance measurements at the determined readout frequency of 2 kHz were considered. The measurement of day O (of an empty chip) before adding cells, was subtracted from all subsequent measurements. We observed variation over time in both chips of the measured impedance of the bottom electrode pair (E1-E4), which can be attributed to the changes in the system (e.g. temperature or conductivity change). To correct for such changes of the system, we introduced the correction factor taking the impedance measured at the bottom compartment and normalizing it to the impedance measured at the bottom compartment at day 0 (Formula 1 and 2). Using the correction factor, for the blank chips without cells, a stable, vertical line around 0 © is observed over a measuring period of 7 days as expected, with an average standard deviation of +/- 244.4 Q which is four times smaller than without correction factor. The effect of the correction factor is also shown for all separate blank chips and chips with Caco2 cells.

The difference in corrected impedance [Zcorrecteal measured between chips with and without Caco2 cells was determined The |Zcoreeteal kept increasing during all 21 days of culture following a similar trend for both chips and reached max value of 13 kQ for the chip 1 and 9.3 kQ for the chip 2. TEER values in literature are often presented in Qcm? in Transwell studies.

We expressed the measured barrier function in Q, because due to the placement of the electrodes not the whole membrane culture area is probed.

The TEER in Qcm? can be estimated by multiplying the results in Q by 0.04 cm?, which is the estimated culture area which is probed by the electrodes.

The calculated TEER values in (Qcm?) only slightly increased from 0 to 50 (chip 1) or 100 (chip 2) at day 10 and successively increased to about 500 or 400 for chip 1 and 2, respectively.

TEER values for Caco2 cells cultured in Transwell systems show varying absolute values of TEER in Qcm?. In the literature gradual increase in cell barrier (or TEER) for the first 20 days in a Transwell study with Caco2 cells are reported, up to a TEER of approximately 450 Qcm?, which is similar as we see in our culture.

Barrier disruption and recovery To test whether the electrodes are sensitive to the changes in the system and to verify that the measured resistance was related to the formation of the tight junctions, the cell monolayer was disrupted by a S mM EGTA treatment for 45 mins.

EGTA is a strong Ca”? chelator, which will affect both adherens junctions and tight junctions, resulting in a disrupted barrier function of the tissue.

The measured impedance decreased to approximately 30.6% {average of the two chips) with respect to day 19 before adding EGTA, indicating a loss of barrier function.

Subsequent incubation with DMEM, providing calcium ions to recover the cellular junctions and thus the barrier function, the measured impedance reached overnight to more than 100%. This indicated a full recovery of the barrier function overnight.

Conclusion A cleanroom-free, versatile fabrication method for the integration of platinum electrode wires in a PDMS OoC allowed us to monitor the barrier function in real-time using impedance spectroscopy.

We monitored and observed the formation of the cell barrier during a 21-day cell culture, as well as disruption of the cell-barrier in response to a treatment with 5 mM EGTA with recovery of the barrier in DMEM.

The proposed method and configuration provide abundant design freedom and the possibility to multiplex both the electrodes and the number of OoCs in one PDMS device.

The method can be applied to monitor the barrier function in a PDMS two-layer OoC device with any kind of cell type forming a monolayer or tissue barrier.

Claims

Conclusions:

A system (1000) comprising a microfluidic device (1), wherein the microfluidic device (1) comprising a first side (2), a second side (3), a device plane (6) between the first side (2) and the second side (3), a first channel (100) having a first channel inlet (101) and a first channel outlet (102), and a first side chamber (150) extending from the first channel (100) between the first channel inlet (101) and the first IO channel outlet (102) in a direction parallel to the device plane (6); wherein the first side chamber (150) is accessible through a first opening (151) either through the first side (2) or through the second side (3); and wherein the system (1000) further comprises a first electrode element (8) configured at least partially in the first opening (151) and the first side chamber (150).

The system (1000) of claim 1, wherein the first electrode element (8) does not extend into the first channel (100).

The system (1000) of any preceding claim, wherein the microfluidic device (1) comprises two or more first side chambers (150), (1) wherein each first side chamber (150) is accessible through a respective first opening (151). ) through the first side (2) or (11) wherein each first side chamber (150) is accessible through a respective first opening (151) through the second side (3).

The system (1000) of any preceding claim, wherein the microfluidic device (1) further includes (i) a second channel (200) and (11) a second side chamber (250) extending from the second channel (200). in a direction parallel to the device plane (6); wherein the second channel (200) is in fluid communication with the first channel (100); and wherein the second side chamber (250) is accessible through a second opening (251) either through the first side (2) or through the second side (3); wherein the first channel (100) has a first channel axis (105) and wherein the second channel (200) has a second channel axis (205), the first channel axis (105) configured parallel to the second channel axis (205), the first channel (100 ) and the second channel (200) together define a microfluidic dual channel (10) having a dual channel axis (15); the microfluidic device (1) further comprising a porous membrane (300) adapted to physically separate the second channel (200) from the first channel (100), the porous membrane (300) being parallel to the device plane (6 ) is arranged with the second channel (200) being in fluid communication with the first channel (100) through the porous membrane (300).

The system (1000) of claim 4, wherein the microfluidic device (1) comprises two or more second side chambers (250), wherein each second side chamber (250) is accessible through a respective second opening (251) or through the first side. (2) or via the second side (3).

The system (1000) of any one of claims 4 to 5, wherein the microfluidic device (1) has n first channels (100), n second channels (200), m first side chambers (150) and p second side chambers (250). wherein n > 2, m > n and p > n, wherein - the first channels (100) are arranged in a plane parallel to the device plane (6), - from each of the first channels (100) at least one individual first side chamber (150) extending in a direction parallel to the device plane (6), and from each of the second channels (200) at least one individual second side chamber (250) extending in a direction parallel to the device plane (6), - each first side chamber (150) is accessible through an individual first opening (151) selected from the first side (2) and the second side (3) and each second side chamber (250) is accessible through an individual second opening (251) or through the single side; and - each of the first channels (100) is configured in fluid communication with a respective second channel (200), the first channel (100) and the second channel (200) together defining a respective microfluidic dual channel (10).

The system (1000) according to any one of the preceding claims 4-6, wherein the microfluidic device (1) (i) has a first layer (19) comprising the one or more first channels (100) and the one or more first side chambers (150) and (11) a second layer (29) comprising the one or more second channels (200) and the one or more second side chambers (250), the first layer (19), the second layer (29) and the optional membrane (300) are at least partially transparent.

The system (1000) of any one of claims 4 to 7, wherein (1) two individual first side chambers (150) extending in opposite directions from the microfluidic dual channel (10) and (ii) two individual second side chambers ( 250) extending in opposite directions from the microfluidic dual channel (10) of at least one of the microfluidic dual channels (10) defining a four-chamber arrangement (45), wherein one of the first side chambers (150) and one of the second side chambers (250 ) extending at a first longitudinal position (21) and another of the first side chambers (150) and another of the second side chambers (250) extending at a second longitudinal position (22), and a distance (L 11) between the first longitudinal position (21) and the second longitudinal position (22) is selected from the range of 0.05-4 mm, and wherein (i) in one of the first side chambers (150) a first electrode element (8 ) at least part configured in the first opening (151) and the first side chamber (150), and (ii) in one of the second side chambers (250) a second electrode element (9) at least partially in the second opening (251) and the second side chamber (250) ) is configured.

A method of manufacturing a system (1000) comprising an IS microfluidic device (1), the method comprising a preparation stage and a bonding stage, wherein - the bonding stage: (i) providing a first layer (19) having a primary first layer side (191) comprising a continuous first recess (192), the first recess (192) comprising a first side extension (154), and (ii) covering the first recess (192) with a cover (290) wherein a first cover side (297) closes the continuous first recess (192) to provide the microfluidic device (1) having a first channel (100) and a first side chamber (150) extending from the first channel (100) includes; - the preparation stage (i) providing a first opening (151) from a secondary first layer side (196) arranged opposite the primary first layer side (191) to the first side extension (154), or (11) providing the first opening (151) from the first cover side (297) to a second cover side (296), which is arranged opposite the first cover side (297), at a location of the first cover side (297) which is used to cover the first side extension ( 154) is configured, wherein the preparation stage is provided before, during and/or after the connection stage.

The method of claim 9, wherein the cover (290) comprises a second layer (29) comprising a primary second layer side (291) including a continuous second recess (292), the second recess (292) having a second side extension (254 )

wherein (i) the first cover side (297) comprises the primary second layer side (291), or (ii) wherein the cover (290) further comprises a porous membrane (300) arranged on the primary second layer side (291), and the first cover side (297) comprises the porous membrane (300); wherein - the bonding stage further comprises connecting the first recess (192) to the second recess (292), optionally via the porous membrane (300), in fluid communication, the first layer (19) the continuous second recess (292) close, defining a second channel (200) and a second side chamber (250) extending from the second channel (200); - the bonding stage further comprising: (i) providing a second opening (251) from the side of the secondary first layer side (196) to the primary first layer side (191), at a location configured to be covered by the second side extension (254) of the cover (290) and further optionally providing the second opening (251) through the optional membrane (300) such that the second opening (251) in the first layer (19) and in the porous membrane (300 ) aligned in the provided microfluidic device (1), or (it) providing the second opening (151) from a secondary second layer side (296) to the second side extension (254); wherein a first recess axis (195) is arranged parallel to a second recess axis (295) when the first recess (192) is fluidly connected to the second recess (292) in the bonding stage, creating a microfluidic dual channel (10 ) comprising a dual channel shaft (15) is provided.

The method of claim 10, wherein the primary first layer side (191) comprises n first recesses (192), each first recess (192) comprising at least one first side extension (154), and the primary second layer side (291) n second recesses (292), wherein each second recess (292) comprises at least one second side extension (254), wherein n > 2; wherein - the joining stage comprises connecting each of the first recesses (192) in fluid communication with an individual second recess (292), the first cover side (297) closing each of the first recesses and the first layer (19) closing each of closes the second recesses whereby (1) n first channels (100), each first channel (100) comprises at least one first side chamber (150) extending from the first channel (100), and (11) n second channels ( 200), wherein each second channel (200) includes at least one second side chamber (250) extending from the second channel (200) being defined;

- the preparation stage comprises: (i) providing a respective first opening (151) from the secondary first layer side (196) to each of the first side extensions (154) and providing a respective second opening (251) from the secondary first layer side ( 196) to the primary first layer side (191) at locations configured to be covered by each of the second side extensions (254) of the cover (290) and further optionally providing the second openings (251) through the optional membrane ( 300), such that the second openings (251) in the first layer (19) and in the porous membrane (300) are aligned in the microfluidic device (1), or (ij) providing the first openings (151) from the first cover side (297) to the second cover side (296) at each of the locations of the cover (290) configured to cover the first side extensions (154) and provide the second openings (251) from the second secondary second layer side (296) to each of the second side extensions (254).

The method of any one of the preceding claims 10-11, wherein the microfluidic device comprises a first side (2) and a second side (3), the first side being arranged closest to the secondary first layer side (196) and the second side (3) is arranged closest to the secondary second layer side (296), wherein in the preparation stage: - the first opening (151) in each first side chamber (150) is configured for access to the first opening (150) through a single side selected from the first side (2) and the second side (3), and - the second opening (251) in each second side chamber (250) is configured to access the second opening (251) through the single side.

The method according to any one of the preceding claims 10-12, wherein the method further comprises - providing at least partially a first electrode element (8) in at least one of the first openings (151) and the respective first side chamber (150) wherein the first electrode element (8) does not extend into the first channel (100); and - providing at least partially a second electrode element (9) in at least one of the second openings (251) and the respective second side chamber (250), wherein the second electrode element (9) does not extend into the second channel (200).

The method according to any one of the preceding claims 10-13, wherein the method comprises: - casting a first polymeric material (11) into a first mold (18) configured to provide the first layer (19) and curing of the first polymeric material (11) in the first mold (18) to provide the first layer (19), and - casting a second polymeric material (21) into a second mold (28) configured to provide the cover (290) and curing the second polymeric material (21) in the second mold (28) to provide the cover (290).

A method for analyzing one or more of a biological cell (2001) and biological tissue (2000), the method comprising: - providing a system (1000) according to any one of the preceding claims 1-8, or obtainable via the method according to any one of claims 9-14, - providing a biological cell to at least one first channel (100), wherein at least one first side chamber (150) comprising or operably coupled to a first electrode element ( 8) extending from the at least one channel (100), - applying an electrical signal to the at least one first channel (100) and measuring a transformed electrical signal at the first electrode element (8), - determining a property of the biological cell based on the electrical signal and the transformed electrical signal.

The method according to claim 15, wherein the method comprises: - providing the system (1000), wherein the microfluidic device (1) comprises at least one microfluidic dual channel (10), wherein a first electrode element (8) is configured in at least one first side chamber (150) extending from the microfluidic dual channel (10) and a second electrode element (9) configured in at least one second side chamber (250) extending from the microfluidic dual channel (10); - growing biological tissue (2000) in one or more of the first channel (100) and the second channel (200) of the at least one microfluidic dual channel (10); - applying the electrical signal to the first electrode element (8) and to the second electrode element (9), and measuring a transformed electrical signal to the first electrode element (8) and the second electrode element (9) to provide a transepithelial electrical resistance (TEER) measurement of the biological tissue (2000) over time.