Method and system for maintaining a controlled dissolved oxygen concentration in a fluidic chip

Abstract

The invention relates to a fluidic chip (101) comprising: - a first slide (100), the first slide (100) being made in a first material having an oxygen permeability of 10-9 cm3(STP).cm.cm-2.s-1.cmHg-1 or less at 25°C; - a second slide (104) comprising a channel (106), the second slide (104) being made in a second material having an oxygen permeability of 10-9 cm3(STP).cm.cm-2.s-1.cmHg-1 or less at 25°C; - a first adhesive layer (114) joining the first slide (100) to the second slide (104), the first adhesive layer (114) being made in a first adhesive composition having an oxygen permeability of 10-9 cm3(STP).cm.cm-2.s-1.cmHg-1 or less at 25°C.

Description

[0001] METHOD AND SYSTEM FOR MAINTAINING A CONTROLLED DISSOLVED OXYGEN CONCENTRATION IN A FLUIDIC CHIP

[0002] Technical field

[0003] The present invention relates to a fluidic chip, an assembly incorporating the fluidic chip, and a method of using the fluidic chip, wherein a controlled concentration in dissolved oxygen is achieved in the fluidic chip.

[0004] Technical background

[0005] In the last two decades, microfluidics has developed into a versatile platform technology, however in most fields the oxygen concentration on chip is an often-overlooked aspect. In biology, reproducing oxygen levels in in vitro systems aims to mimic in vivo cellular gaseous microenvironments which drastically vary among organs.

[0006] To produce on-chip oxygen-sensitive chemical reactions in a predictable and reproducible manner, oxygen concentrations must be precisely controlled on chip. In the case of oxygen sensitive compounds (such as Grignard reagents) and reactions (such as metal-mediated cross-couplings), oxygen should be carefully removed from the medium to avoid the formation of undesired products or the degradation of the starting material. In the field of chemistry in batch, different methods can be used to achieve low concentration in oxygen such as inertization manifolds, inert gas purging or freeze-pump-thaw techniques.

[0007] Controlling dissolved oxygen level in microfluidic chips is challenging and two methods are mainly used: the inclusion of a gaseous channel or the inclusion of oxygen scavengers. The first solution relies on gas diffusion through a thin polydimethylsiloxane (PDMS) membrane from a side channel close to the channel of interest. Using this method, as disclosed in the article, “Microfluidic platform for three-dimensional cell culture under spatiotemporal heterogeneity of oxygen tension” by Koens et al., 2020, DOI: 10.1063 / 1.5127069, it is possible to reach 0.3% of oxygen in 15 min. However, using this method, reoxygenation can occur through the PDMS and the chip must be perfused constantly with gas which consume gas bottle at a fast rate. Other disadvantages of this method are that the liquid can evaporate through the diffusion membrane. The second most common method for on-chip oxygen control relies on chemical induction. Different scavenging agents can be used such as pyrogallol or sodium sulfite. However, such a method requires a constant perfusion of chemicals to maintain a constant concentration of the reagent.

[0008] Another method recently described by Bouquerel et al. in “Precise and fast control of the dissolved oxygen level for tumor-on-chip”, 2022, DOI: 10.1039 / D2LC00696K, as well as in WO 2022 / 069662, allows a fine control of the dissolved oxygen level in the cell culture medium perfused in a microfluidic chip, through the simultaneous regulation of the gas composition and the inlet reservoir pressure by modulating the pneumatic valve opening. This method offers unprecedented features such as an oxygen equilibration time lower than 3 minutes and an accuracy of 3 mmHg.

[0009] The most common microfluidic chip material is PDMS, but it has been found by the present inventors that microfluidic chips made of PDMS do not allow an effective control of oxygen concentration in the chip.

[0010] Glass is another known material for microfluidic chips. Fabrication processes on glass substrates, however, are time-consuming and clean-room facilities and / or specific microfabrication equipment are required.

[0011] To fabricate various patterns on glass plates, there are several typical techniques available such as etching, laser-ablation, mechanical erosion, electrical process, embossing or additive fabrication with liquid glass. Usual glass- to-glass bonding techniques include anodic bonding (at high voltage or high temperature), surface activated bonding, fusion bonding (at high temperature) or laser welding. The resulting microchips are therefore usually expensive for use as disposable devices. Moreover, a high temperature bonding process can lead to channel deformation and is not compatible with the inclusion of sensors, such as oxygen sensors, pH sensors or others. The inclusion of oxygen sensors in a hermetic microfluidic chip enabling precise oxygen control has broad applications in various fields. For instance, in biology, such a microfluidic chip could be utilized as a cell respirometer.

[0012] The measurement of cellular respiration is among the most fundamental experimental techniques that have formed the basis of our understanding of bioenergetics. The gold standard method is a multiwell plate-based assay platform (XF24 ExtracellularFlux Analyzer, SeaHorse) which uses fluorescent sensors close to the cells in a chamber with modulable volume. However, such plastic plate-based respirometers are susceptible to a constant entrance of O2 from the environment. Therefore, a correction algorithm has been developed to deconvolute the biological oxygen consumption rate from the measured O2. Such post-measurement correction procedure is however undesirable.

[0013] Document AU 2020369940 A1 relates to cell culture systems and methods. The cell culture systems are configured with interchangeable cartridges. The systems are configured to have multiple connected cell culture chambers. The document also relates to gas-impermeable cell culture chambers and methods for generating cells in closed systems and methods for recycling cell culture medium.

[0014] The article, “Multi-layer microfluidic glass chips for microanalytical applications”, by Daridon et al. and published 27thJuly 2001 , DOI 10.1007 / s002160101004, relates to microfluidic devices made entirely from glass, for use with reagents which would prove highly corrosive for silicon. Chips consist of three layers of glass wafers bonded together by fusion bonding. On the inside wafer, a network of microfluidic channels is created by photolithography and wet chemical etching. Low dead-volume fluidic connections between the layers are fabricated by spark-assisted etching (SAE).

[0015] The article, “A simple approach to fabricate multi-layer glass microfluidic chips based on laser processing and thermocompression bonding”, by Han et al. and published 16thAugust 2021 , https: / / doi.org / 10.1007 / s10404-021-02479-y, relates to a fabrication process based on rapid laser cutting of single glass layers and one-time thermocompression bonding of multi-layer chips and demonstrated it with a five-layer microfluidic chip for three-dimensional (3D) hydrodynamic focusing.

[0016] The article, “Microfluidics in glass: technologies and applications”, by Iliescu et al., MIDEM 2006 Conference Slovenia, relates to technological aspects of patterning and assembling of glass wafers for microfluidic applications as well as three applications of the developed technologies. Special masking layers (Cr / Au / Photoresist and a:Si / SiC / Photoresist) were designed, fabricated and tested for deep wet etching of glass.

[0017] The article, “An all-glass 12 pm ultra-thin and flexible microfluidic chip fabricated by femtosecond laser processing” by Yalikun et al. and dated 2016 relates to a method, using femtosecond laser processing, to fabricate a 100%- glass-based 12 pm ultra-thin and flexible micro-fluidic chip.

[0018] The article, “A glass microfluidic chip adhesive bonding method at room temperature”, by Pan et al., published 7thNovember 2006, DOI 10.1088 / 0960- 1317 / 16 / 12 / 020, relates to a method using UV epoxy resin for the bonding of glass blanks and patterned plates at room temperature. However, the oxygen permeability of the epoxy resin was not investigated as well as the hermeticity of the complete assembled chip. Within this context, there is still a need for a fluidic chip which is easy to manufacture, and which can be fed with a liquid having a controlled concentration in dissolved oxygen, in such a way that an oxygen concentration target value in the fluidic chip can be rapidly achieved, and that an oxygen concentration target value in the fluidic chip can be maintained after stopping the inflow of fresh liquid.

[0019] Summary of the invention

[0020] The invention relates to the following items:

[0021] Item 1 . A fluidic chip comprising:

[0022] - a first slide, the first slide being made in a first material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;

[0023] - a second slide comprising a channel, the second slide being made in a second material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;

[0024] - a first adhesive layer joining the first slide to the second slide, the first adhesive layer being made in a first adhesive composition having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

[0025] Item 2. The fluidic chip according to item 1 , wherein the channel is an area of the second slide having a reduced thickness relative to the rest of the second slide.

[0026] Item 3. The fluidic chip according to item 1 , wherein the channel is a through hole within the second slide.

[0027] Item 4. The fluidic chip according to item 3, further comprising:

[0028] - a third slide, the third slide being made in a third material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;

[0029] - a second adhesive layer joining the third slide to the second slide, the second adhesive layer being made in a second adhesive composition having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

[0030] Item 5. The fluidic chip according to item 4, wherein the second adhesive composition is the same as the first adhesive composition.

[0031] Item 6. The fluidic chip according to any one of items 1 to 5, wherein:

[0032] - the first material has an oxygen permeability of 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C; and / or - the second material has an oxygen permeability of

[0033] 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C; and / or

[0034] - the third material, if present, has an oxygen permeability of

[0035] 10’1° cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C; and / or

[0036] - the first adhesive layer has an oxygen permeability of 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C; and / or

[0037] - the second adhesive layer, if present, has an oxygen permeability of 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

[0038] Item 7. The fluidic chip according to any one of items 1 to 6, wherein the first material, the second material, and the third material, if present, are the same.

[0039] Item 8. The fluidic chip according to any one of items 1 to 7, wherein the first material is glass or fused silica; and / or the second material is glass or fused silica; and / or the third material, if present, is glass or fused silica.

[0040] Item 9. The fluidic chip according to any one of items 1 to 8, wherein the first adhesive composition and / or the second adhesive composition, if present, comprises a cyclic olefin copolymer (COC), an epoxy resin, an acrylate resin, a polyamide, polyethylene terephthalate, polyethylene naphthalate, ethylene vinyl alcohol, polyvinyl chloride, polyvinylidene chloride, liquid crystal polymers and combinations thereof.

[0041] Item 10. The fluidic chip according to any one of items 1 to 9, wherein the first adhesive composition and / or the second adhesive composition, if present, is a pressure sensitive, temperature sensitive, and / or light sensitive adhesive, and is preferably a UV light sensitive adhesive.

[0042] Item 11 . The fluidic chip according to any one of items 1 to 10, wherein the first adhesive layer and / or the second adhesive layer, if present, has a thickness of 200 pm or less, preferably of 150 pm or less, more preferably of 100 pm or less.

[0043] Item 12. The fluidic chip according to any one of items 1 to 11 , wherein the first adhesive layer and / or the second adhesive layer, if present, extends in contact with the second slide over a distance from the channel of from 1 mm to 50 mm, preferably from 2 mm to 20 mm, more preferably from 5 mm to 15 mm.

[0044] Item 13. The fluidic chip according to any one of items 1 to 12, wherein the first adhesive layer and / or the second adhesive layer, if present, substantially fully covers the second slide except the channel.

[0045] Item 14. The fluidic chip according to any one of items 1 to 13, comprising at least two connection ports for the channel, each connection port comprising a hole in the first glass slide.

[0046] Item 15. The fluidic chip according to any one of items 1 to 14, comprising a sensor in the channel, the sensor being preferably selected from an optical sensor, a pressure sensor, a temperature sensor, a concentration sensor, a flow sensor, a light sensor, a camera, or a current or voltage sensor.

[0047] Item 16. An assembly, comprising:

[0048] - the fluidic chip according to any one of items 1 to 15;

[0049] - a feeding device fluidically connected to the fluidic chip, the feeding device configured for feeding liquid medium having a controlled concentration in dissolved oxygen to the fluidic chip.

[0050] Item 17. The assembly of item 16, wherein the feeding device is configured for feeding liquid medium having a partial pressure of oxygen of less than 150 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg.

[0051] Item 18. The assembly of item 16 or 17, wherein the feeding device comprises at least one feeding module, and each feeding module comprises:

[0052] - a liquid reservoir containing a liquid medium and provided with a gas inlet, a gas outlet and a liquid outlet,

[0053] - an admission line configured to provide a gaseous medium into the liquid reservoir, the admission line being connected to the gas inlet, and

[0054] - an emission line configured to withdraw the gaseous medium from the liquid reservoir, the emission line being connected to the gas outlet;

[0055] - at least one control valve associated with the admission line(s) and at least one control valve associated with the emission line(s).

[0056] Item 19. The assembly according to any one of items 16 to 18, wherein the fluidic chip is constructed so that the time taken to reach a predetermined oxygen partial pressure of 15 mmHg within the channel from ambient oxygen pressure conditions, is less than 5 minutes, when liquid medium having an oxygen partial pressure of 15 mmHg is perfused to the fluidic chip at a flow rate of 100 pL / min.

[0057] Item 20. The assembly according to any one of items 16 to 19, wherein the fluidic chip is constructed so that the time taken to reach ambient oxygen pressure conditions from a partial pressure of oxygen of 15 mmHg within the channel is greater than 2 hours, preferably greater than 10 hours, in the absence of perfusion of liquid medium within the channel.

[0058] Item 21 . A method of feeding liquid medium to the fluidic chip according to any one of items 1 to 15, comprising a step of providing liquid medium having a controlled concentration in dissolved oxygen, and a step of flowing said liquid medium having a controlled concentration in dissolved oxygen to the fluidic chip.

[0059] Item 22. The method of item 21 , the liquid medium having a controlled concentration in dissolved oxygen has a partial pressure of oxygen of less than 150 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg.

[0060] Item 23. The method of item 21 or 22, comprising a step of stopping the flow of liquid medium to the fluidic chip and retaining the liquid medium within the fluidic chip.

[0061] Item 24. The method of any one of items 21 to 23, which is a method for culturing a living entity in the fluidic chip.

[0062] Item 25. A method for manufacturing the fluidic chip according to any one of items 1 to 15, the method comprising:

[0063] - bonding the first slide with the second slide owing to the first adhesive layer;

[0064] - optionally bonding the second slide with the third slide owing to the second adhesive layer, if present.

[0065] Item 26. The method according to item 25, comprising preliminary steps of forming the channel into the second slide and / or forming at least two holes into the first slide.

[0066] Item 27. An assembly, comprising:

[0067] - a fluidic chip comprising a first slide made in a first material, a second slide made in a second material and comprising a channel, and a first adhesive layer made in a first adhesive composition and joining the first slide to the second slide;

[0068] - a feeding device fluidically connected to the fluidic chip, the feeding device configured for feeding liquid medium having a controlled concentration in dissolved oxygen to the fluidic chip; wherein the fluidic chip is constructed so that the time taken to reach a predetermined oxygen partial pressure of 15 mmHg within the channel from ambient oxygen pressure conditions is less than 5 minutes, when liquid medium having an oxygen partial pressure of 15 mmHg is perfused to the fluidic chip at a flow rate of 100 pL / min; and / or wherein the fluidic chip is constructed so that the time taken to reach ambient oxygen pressure conditions from a partial pressure of oxygen of 15 mmHg within the channel is greater than 2 hours, preferably greater than 10 hours, in the absence of perfusion of liquid medium within the channel.

[0069] Item 28. The assembly of item 27, wherein the feeding device is configured for feeding liquid medium having a partial pressure of oxygen of less than 150 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg. Item 29. The assembly of item 27 or 28, wherein the feeding device comprises at least one feeding module, and each feeding module comprises:

[0070] - a liquid reservoir containing a liquid medium and provided with a gas inlet, a gas outlet and a liquid outlet,

[0071] - an admission line configured to provide a gaseous medium into the liquid reservoir, the admission line being connected to the gas inlet, and

[0072] - an emission line configured to withdraw the gaseous medium from the liquid reservoir, the emission line being connected to the gas outlet;

[0073] - at least one control valve associated with the admission line(s) and at least one control valve associated with the emission line(s).

[0074] Item 30. The assembly of any of items 27 to 29, wherein the channel is an area of the second slide having a reduced thickness relative to the rest of the second slide.

[0075] Item 31 . The assembly of any of items 27 to 29, wherein the channel is a through hole within the second slide.

[0076] Item 32. The assembly according to item 31 , further comprising:

[0077] - a third slide made in a third material;

[0078] - a second adhesive layer made in a second adhesive composition and joining the third slide to the second slide.

[0079] Item 33. The assembly according to item 32, wherein the second adhesive composition is the same as the first adhesive composition.

[0080] Item 34. The assembly of any of items 27 to 33, wherein the first material, the second material, and the third material, if present, are the same.

[0081] Item 35. The assembly of any of items 27 to 34, wherein the first material is glass or fused silica; and / or the second material is glass or fused silica; and / or the third material, if present, is glass or fused silica.

[0082] Item 36. The assembly of any of items 27 to 35, wherein the first adhesive composition and / or the second adhesive composition, if present, comprises a cyclic olefin copolymer (COC), an epoxy resin, an acrylate resin, a polyamide, polyethylene terephthalate, polyethylene naphthalate, ethylene vinyl alcohol, polyvinyl chloride, polyvinylidene chloride, liquid crystal polymers and combinations thereof.

[0083] Item 37. The assembly of any of items 27 to 36, wherein the first adhesive composition and / or the second adhesive composition, if present, is a pressure sensitive, temperature sensitive, and / or light sensitive adhesive, and is preferably a UV light sensitive adhesive. Item 38. The assembly of any of items 27 to 37, wherein the first adhesive layer and / or the second adhesive layer, if present, has a thickness of 200 pm or less, preferably of 150 pm or less, more preferably of 100 pm or less.

[0084] Item 39. The assembly of any of items 27 to 38, wherein the first adhesive layer and / or the second adhesive layer, if present, extends in contact with the second slide over a distance from the channel of from 1 mm to 50 mm, preferably from 2 mm to 20 mm, more preferably from 5 mm to 15 mm.

[0085] Item 40. The assembly of any of items 27 to 39, wherein the first adhesive layer and / or the second adhesive layer, if present, substantially fully covers the second slide except the channel.

[0086] Item 41. The assembly of any of items 27 to 40, comprising at least two connection ports for the channel, each connection port comprising a hole in the first glass slide.

[0087] Item 42. The assembly of any of items 27 to 41 , comprising a sensor in the channel, the sensor being preferably selected from an optical sensor, a pressure sensor, a temperature sensor, a concentration sensor, a flow sensor, a light sensor, a camera, or a current or voltage sensor.

[0088] The present invention makes it possible to address the need mentioned above. In particular, the invention provides for a fluidic chip which is easy to manufacture, and which can be fed with a liquid having a controlled concentration in dissolved oxygen, in such a way that an oxygen concentration target value in the fluidic chip can be rapidly achieved, and that an oxygen concentration target value in the fluidic chip can be maintained after stopping the inflow of fresh liquid

[0089] This fluidic chip has improved properties for example for culturing living entities or carrying out chemical reactions at a controlled dissolved oxygen concentration.

[0090] This is achieved by the fact that the chip is made from an assembly of layers, including one or more adhesive layers, wherein the layers including the adhesive layer(s) have a sufficiently low oxygen permeability, therefore substantially preventing oxygen transfer between the interior of the fluidic chip and ambient air.

[0091] This multilayer structure simplifies the manufacturing of the fluidic chip, as the slides can simply be stacked upon one another and joined together by an adhesive composition, which does not require any involvement of a clean room or any special microfabrication equipment. The method for manufacturing the chip is therefore both user-friendly and easy to implement in non-specialized laboratories. This in turn allows for the chip to be a low-cost chip that is quick to assemble. The use of adhesive composition(s) makes it possible to avoid using specialized and complicated procedures such as anodic bonding, surface activated bonding, fusion bonding, or laser welding that can even result in deformation of chip features and / or architecture (e.g. high temperature bonding).

[0092] Brief description of the drawings

[0093] Non-limiting examples will now be described in reference to the accompanying drawings, where:

[0094] FIG. 1A-1 D show an example of the fluidic chip and its respective components. FIG. 1 A and FIG. 1 B show examples of the first slide and the second slide respectively. FIG. 1 C shows an example of the fluidic chip in an intermediate assembling state. FIG. 1 D shows an example of the fluidic chip once assembled.

[0095] FIG. 2A shows the same fluidic chip in an exploded view.

[0096] FIG. 2B shows the same fluidic chip, with some parts removed.

[0097] FIG. 3 shows an example of a manufacturing process flow for the microfluidic chip.

[0098] FIG. 4 displays an example of an assembly comprising the fluidic chip and a feeding device for feeding the fluid chip with liquid medium.

[0099] FIG. 5 shows an example of finite element analysis run on a microfluidic chip as described herein. The graphs illustrate a deoxygenation process. Each graph illustrates, for different flow rates, oxygen partial pressure (pO2 (mmHg)) as a function of time (min). See example 1 .

[0100] FIG. 6 shows an example of finite element analysis run on the microfluidic chip. The graphs illustrate a reoxygenation process. Oxygen partial pressure (pO2 (mmHg)), in the channel is plotted as a function of time (h). See example 2.

[0101] FIG. 7A shows examples of finite element analysis run on the microfluidic chip. The graphs illustrate a deoxygenation process (left) and a reoxygenation process (right). Oxygen partial pressure (pO2 (mmHg)) in the channel is plotted as a function of time (min in the left hand side graph, hours on the right hand side graph). See example 3.

[0102] FIG. 7B shows examples of finite element analysis run on the microfluidic chip. The graphs illustrate a deoxygenation process (left) and a reoxygenation process (right). Oxygen partial pressure (pO2 (mmHg)) in the channel is plotted as a function of time (min in the left hand side graph, hours on the right hand side graph). See example 3.

[0103] FIG. 8 relates to experimental tests showing: in the top graph, the time needed to reach a target of 16.5 mmHg (in min) as a function of flowrate of chip perfusion (pL / min, indication on the X-axis) for different fluidic chips; and in the bottom graph, the increase in oxygen partial pressure (in mmHg) after reaching the target partial pressure and ceasing the flow of liquid medium having a controlled concentration in oxygen for 2 hours for different fluidic chips. See example 4.

[0104] FIG. 9 shows the partial pressure of oxygen (in mmHg) experimentally measured in a fluidic chip as described herein, as a function of time (in min). The fluidic chip contains living cells. The arrows indicate the inflow of liquid medium having a certain partial pressure of oxygen. Between the arrows, the flow of liquid medium was stopped. See example 5.

[0105] Detailed description

[0106] The invention will now be described in detail without limitation in the following description.

[0107] Fluidic Chip

[0108] By “fluidic chip" is meant a chip comprising at least one channel wherein liquid medium can flow. The fluidic chip can be millifluidic, microfluidic or nanofluidic. Preferably, it is a microfluidic chip.

[0109] By “millifluidic" is meant a system in which the minimal channel dimensions are of the order of 1 -10 mm. By “microfluidic" is meant a system in which the minimal channel dimensions are of the order of 1 to less than 1000 pm. By “nanofluidic" is meant a system in which the minimal channel dimensions are of the order if less than 1 pm.

[0110] It is provided a fluidic chip which comprises a multilayer structure.

[0111] The fluidic chip comprises at least three layers, namely a first slide, a second slide, and a first adhesive layer joining the first slide to the second slide. The multilayer structure may consist of these three layers.

[0112] Alternatively, the fluidic chip may comprise five layers, i.e., in addition to the three above layers: a third slide, and a second adhesive layer joining the third slide to the second slide. The multilayer structure may consist of these five layers. Alternatively, the multilayer structure may comprise these five layers as well as additional layers.

[0113] By “layeh is meant a material having a certain thickness. The layers may each have a different thickness from one another.

[0114] By “slide" is meant a thin flat slab of material, for example of rectangular form.

[0115] The second layer comprises a channel. By “channel” is meant a passage, in which a liquid medium can flow. The channel may be an internal chamber once the chip is assembled. The channel may have a substantially longitudinal shape.

[0116] The first slide, second slide and third slide (if present) are respectively made in a first material, second material and third material.

[0117] The first adhesive layer and the second adhesive layer (if present) are respectively made in a first adhesive composition and second adhesive composition. The first adhesive composition and second adhesive composition are different from the first, second and third materials.

[0118] By “adhesive composition" is meant a substance applied to one or both surfaces of two separate items that binds them together and resists their separation. The adhesive composition may be applied under ambient conditions (i.e. does not have to be heated). The adhesive composition may be a non- metallic composition.

[0119] By “joins" is meant bonds or connects fixedly, for example after curing of the adhesive composition.

[0120] FIG. 1 A to 1 D and 2A illustrate an embodiment of a fluidic chip comprising a five-layer assembly.

[0121] In FIG. 1 A to 1 D, direction Z is the direction of the thickness of the slides. Directions X and Y are perpendicular to direction Z and are perpendicular to each other. For example, if the slides are rectangular in shape, direction X can be the direction of the width, while direction Y can be the direction of the length.

[0122] FIG. 1 D shows an example of a fluidic chip 101 , shown in cross-section in the YZ direction. The chip 101 comprises five layers: a first slide 100, a first adhesive layer 114, a second slide 104, a second adhesive layer 116, and a third slide 108. The first adhesive layer 114 joins the first slide 100 to the second slide 104, and second adhesive layer 116 joins the second slide 104 to the third slide 108. The first, second and third slides 100, 104, 108 may have substantially the same thickness. Alternatively, the second slide 104 may have a different thickness to the other slides 100, 108. The thickness of the second slide 104 may be dependent on the desired thickness of the channel 106. Each of the adhesive layers 114, 116 may have substantially the same thickness. The thickness of each of the adhesive layers 114, 116 may be less than the thickness of each of the slides 100, 104, 108.

[0123] Each of the slides 100, 104, 108 may be stacked evenly upon one another. This can assist in ensuring the exact desired positioning of the channel 106 in the chip 101. The channel 106 may be located towards (or at) the center of the chip 101 in the X direction (FIG. 1 C). The channel 106 may be located towards (or at) the center of the chip 101 in the Y direction. The channel 106 may extend along the Y direction.

[0124] The third slide 108 is preferably intact, i.e. devoid of channels or holes, i.e. neither patterned nor drilled.

[0125] As shown in FIG. 1 B, 1 C and 1 D, the second slide 104 comprises a single channel 106. Alternatively, the second slide 104 may comprise multiple channels 106.

[0126] The channel 106 may be of a rectangular shape. Alternatively, the channel 106 may have a different shape; for example the width of the channel 106 may vary along the length thereof. The channel 106 may be in the form of one straight conduit. Alternatively, the channel 106 may have a more complex shape. The channel may comprise one or more curved portions(s), and / or may comprise one ore more linear portion(s) having different orientations. The channel 106 may be in the form of a conduit comprising one or more branches or sub-conduits. This can be advantageous depending on the type of test to be performed within the channel 106.

[0127] The fluidic chip 101 is preferably devoid of separate gas flow elements. Thus, the chip 101 may be devoid of a gas channel, or a gas chamber or a gas- permeable material (such as a membrane). This makes it possible to reduce fabrication cost and simplify the fabrication process.

[0128] As can be seen in FIG. 1 B the channel 106 may be formed by a through hole in the second slide 104.

[0129] The chip 101 may comprise two or more liquid inlets and / or outlets for each channel 106. These are formed by holes 102a, 102b in the first slide 100. Each hole 102a, 102b may be positioned in the first slide 100 so that each hole 102a, 102b is directly above a respective location of the channel 106. As shown in FIG. 1 C and FIG. 1 D, the first hole 102a may be directly over one end of the channel 106 and the second hole 102b may be directly over an opposite end of the channel 106. In this way, a liquid medium can be perfused through one hole 102a and may flow out the other hole 102b.

[0130] The chip 101 may comprise fluidic connectors 112 fixed on the first slide 100 and associated with the respective holes 102a, 102b, as shown in FIG. 1 D. As disclosed in FIG. 4 (discussed in more detail below), the chip 101 may be connected to one or more flow lines 213, 214 (each flow line may be for example a fused silica capillary). One connector 1 12 may serve as a connection port to a flow line 213 that may provide a liquid medium to the channel 106. The other connector 112 may serve as a connection port to another flow line 214 that may receive the liquid medium flowing out of the channel 106. Each connector 112 may be in fluid communication with a respective hole 102a, 102b so as to provide an access for the liquid medium to the channel 106 and an exit for the liquid medium from the channel 106. Each connector 112 may be fixed to the first slide 100 with an adhesive, for example using an epoxy adhesive (e.g. Araldite® 2000).

[0131] Each of the slides 100, 104, 108 may have the same dimensions. Each of the slides 100, 104, 108 may have the same length (i.e. in the Y direction) and the same width (i.e. in the X direction). Such dimensions may facilitate the aligning and may result in the edges of the three slides 100, 104, 108 being flush with one another.

[0132] The channel 106 may be at a minimum distance from each respective edge of the chip 101 (i.e. in both X and Y directions), the distance being preferably at least 2 mm, more preferably at least 5 mm; and ranging for example from 2 mm to 20 mm, preferably from 5 mm to 15 mm. Ensuring a minimum distance between the channel and the edges of the chip 101 may improve the control of dissolved oxygen concentration within the channel 106.

[0133] If the channel 106 has a substantially linear, longitudinal shape, as shown in FIG. 1 B, the channel 106 may be closer to the edges of the chip 101 in the X direction than in the Y direction.

[0134] The adhesive layers 114, 116 can not only act as a means for joining (or bonding) the slides 100, 104, 108 together, but also as sealing layers between the channel 106 and the external environment (i.e. outside the chip 101 ). By joining the slides 100, 104, 108 together, the adhesive more accurately seals the slides 100, 104, 108 of the chip 101 than the likes of tape.

[0135] The slides 100, 104, 108 may be rectangular shaped slides. Each slide 100, 104, 108 may have a length ranging from 50 mm to 100 mm, for example ranging from 60 mm to 90 mm, for example ranging from 70 mm to 80 mm. Each slide 100, 104, 108 may have a width ranging from 10 mm to 50 mm, for example ranging from 10 mm to 40 mm, for example ranging from 15 mm to 35 mm, for example ranging from 20 mm to 30 mm. Each slide 100, 104, 108 may have a thickness of at least 100 pm, or at least 200 pm, and preferably ranging from 0.5 mm to 5 mm, for example ranging from 0.5 mm to 3 mm, for example ranging from 0.5 mm to 2 mm, for example ranging from 0.5 mm to 1.5 mm, for example approximately 1 mm.

[0136] The channel 106 may have a depth that is equal to or less than the thickness of the second slide 104. The channel 106 may have a length ranging from 5 mm to 80 mm, for example ranging from 10 mm to 70 mm, for example ranging from 15 mm to 60 mm, for example ranging from 20 mm to 50 mm, for example ranging from 25 mm to 40 mm, for example ranging from 30 mm to 40 mm. The channel 106 may have a width ranging from 0.5 mm to 20 mm, for ranging example from 0.5 mm to 10 mm, for example from 0.5 to 5 mm, for example ranging from 1 mm to 5 mm, for example from 1 .5 mm to 4.5 mm. It may be preferable for the length of the channel 106 to be greater than the width of the channel 106 so as to form a rectangle, for example the length being 37 mm and the width being 3 mm.

[0137] The chip may comprise one or more sensors 110. Each sensor 110 may be located on the third slide 108. The sensor 110 may be located on the same side of the third slide 108 as that which is joined to the second slide 104 by the second adhesive layer 116. The sensor 110 may therefore be inside the channel 106. Alternatively, the sensor 110 may be located on an external side of the third slide 108, that is to say the sensor 110 may be located on the surface of the third slide 108 that is opposite to the surface of the third slide 108 that is in contact with the second adhesive layer 116. Alternatively, the sensor 110 may be located on the first slide 100, either on the same side as that which is joined to the second slide 104 or on the opposite side. It is also possible to use a sensor embedded in one of the slides 100, 104, 108, or inserted through one of the slides.

[0138] Sensors which may be used in the invention may include optical, optomechanical, electrochemical, electroacoustic, electromechanical or thermoelectric sensors. The sensors may comprise a substrate allowing to capture target molecules. The sensors may include supramolecular sensors. By “supramolecular sensor” is meant a sensor including a molecule having a binding site that is specific for an analyte, wherein the molecule and the analyte form a supramolecular assembly when the analyte binds to the molecule. Analyte binding may be registered through a physical, chemical or biochemical mechanism, such as a colorimetric, fluorescent or electrochemical signal. The binding of the analyte at the binding site may preferably be achieved noncovalently, such as by electrostatic interaction, ionic interaction, cation-Pi interaction, hydrogen bonding, Pi-stacking and van der Waals forces.

[0139] The sensors may comprise cameras or other imaging devices, which may in particular be configured to detect fluorescent markers, such as fluorescent antibodies which may be encapsulated in an external matrix (e.g., via ELISA testing). In particular, such fluorescent markers may be detected directly in the chip 101. In particular, fluorescent markers within cells in the chip 101 may be detected. A sensor may comprise a membrane with fluorescent antibodies. Additionally or alternatively, sensors may include chemosensor arrays, organic field-effect transistors, an analysis chamber and / or ELISA microarrays. In preferred embodiments, the sensor 110 may be an oxygen sensor, such as for example an oxygen optical sensor, a pressure sensor, a temperature sensor, a concentration sensor, a flow sensor, a light sensor, a camera, or a current or voltage sensor. Several of these sensors may be present in the chip.

[0140] As a non illustrated alternative, the fluidic chip can comprise a three-layer assembly rather than a five-layer assembly. In that case, the channel in the second slide may be formed as an area having a reduced thickness relative to the rest of the second slide. This reduced thickness area may be formed for example by chemical etching, laser ablation, mechanical erosion, electrical ablation, embossing or additive fabrication with liquid glass. Other than that, the first slide, second slide and first adhesive layer may be as described above in connection with the five-layer assembly.

[0141] Oxygen permeation

[0142] The materials and adhesive compositions used for making the multilayer structure of the fluidic chip are selected in order to minimize oxygen entry into the channel 106 or exit from the channel 106 from / to the environment.

[0143] More precisely, each of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) has an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

[0144] Therefore, each of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) may have an oxygen permeability of 0 to 10’9cm3(STP).cm.cm’2.s’1.cmHg’1at 25°C.

[0145] In some variations, the oxygen permeability of any of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) is 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C. For example, the oxygen permeability of all of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) is 10’10cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

[0146] In some variations, the oxygen permeability of any of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) is 10’11cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C. For example, the oxygen permeability of all of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) is 10’11cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C. “cm3(STP)” is standard cubic centimeter, which represents the number of gas molecules or moles that would occupy one cubic centimeter at standard temperature and pressure, as calculated via the ideal gas law.

[0147] By way of background information:

[0148] 1 barrer = 1 O’10cm3(STP).cm.cm’2.s’1.cmHg’1;in SI units, 1 barrer = 3.35 x 10-16mol.m.rrr2.s’1.Pa’1.

[0149] Oxygen permeability can be determined based on a measurement of the oxygen transmission rate.

[0150] The oxygen transmission rate can be measured through a film of material, in accordance with ASTM D3985-17 unless otherwise specified. The film may have a size of 10 cm x 10 cm, and a thickness of 2 mm or less. To measure the oxygen transmission rate of the adhesive composition, the adhesive composition is cast as a standalone film, and hardened (e.g. cured) in the same manner as when preparing the adhesive layer(s) of the fluidic chip. Oxygen permeability is obtained by dividing the measured oxygen transmission rate by the difference between the partial pressure of oxygen on the two sides of the film and by multiplying by the thickness of the film.

[0151] The oxygen permeability parameter combines both solubility and diffusion rate in a same value. The oxygen permeability coefficient (Dk) can be expressed as the product of the diffusion coefficient (D) and solubility (k). The diffusion coefficient is the 2-dimensional speed at which oxygen travels through the material. The solubility quantifies the oxygen saturation in the material.

[0152] Each (preferably all) of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) may have an oxygen diffusion coefficient of 10’1° m2 / s or less, preferably of 10’11m2 / s or less, more preferably of 10’12m2 / s or less. The oxygen diffusion coefficient may be for example from 0 to 10’10m2 / s, or from 0 to 10’11m2 / s, or from 0 to 10’12m2 / s.

[0153] The rate of oxygen diffusion is described by Fick’s law, which expresses a linear relationship between the diffusion flux of oxygen and its concentration gradient:

[0154] . dC02dC02

[0155] J = “ — = D * — — Gtt dx with J being the diffusion flux (mol.s’1.rrr2), D the diffusion coefficient (m2.s’1) and being the gradient of oxygen in the material (mol.nr4). D is affected by the size of the permeant and also by the material characteristics, for example in case of a polymer the proportion of crystalline and amorphous regions. Oxygen solubility is described by Henry’s law: C02eq= Kh* pO2with C02eqbeing the concentration in oxygen at equilibrium (mol.rrr3), Khthe Henry’s coefficient (mol.nr3Pa-1) and pO2the partial pressure of oxygen (Pa). The solubility of oxygen in the adhesive is dependent upon the chemical relationship as well as upon the affinity between the oxygen molecule and the material.

[0156] Each (preferably all) of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) may have an oxygen solubility of 2 mol / m3or less, preferably of 1.5 mol / m3or less, more preferably of 1 mol / m3or less, even more preferably of 0.75 mol / m3or less. For example the oxygen solubility may be from 0 to 2 mol / m3, preferably from 0 to 1 .5 mol / m3, more preferably from 0 to 1 mol / m3, even more preferably from 0 to 0.75 mol / m3.

[0157] Each (preferably all) of the first material, second material, third material (if present), first adhesive composition and second adhesive composition (if present) may be made of a non-toxic material, or a material of low cytotoxicity. This is useful when the fluidic chip is issued in biological processes, such as a culture process.

[0158] The first material and second material may be the same or different. Preferably they are the same. The third material (if present) may be the same or different relative to each of the first material and second material. Preferably, the third material is the same as first material and second material.

[0159] The first material, second material, third material are preferably substantially transparent in order to facilitate visual monitoring of the content of the channel.

[0160] The first material, second material and / or third material may in particular be selected from glass, fused silica, and a polymer composition. The polymer composition may comprise or consist of cyclic olefin copolymer (COC), an acrylate resin such as polymethylmethacrylate resin, a polyamide, polyethylene terephthalate, polyethylene naphthalate, ethylene vinyl alcohol, polyvinyl chloride, polyvinylidene chloride and / or a liquid crystal polymer. Glass and fused silica are especially preferred.

[0161] In addition to being highly oxygen-impermeable, glass is also well- characterized and has advantageous physical properties, including high electrical resistivity, high thermal conductivity, and broad light transmissivity.

[0162] The glass slides may be conventional glass slides. This is beneficial next to alternative microfluidic chips, such as those formed using anodic bonding, which requires a special type of glass. The glass may be brittle or substantially inflexible. The glass may be transparent, such that the chip itself is transparent. It may therefore be possible to observe a sample inside the channel, and for example take images of the sample. The glass slide may be an optical glass slide. By way of example, each slide 100, 104, 108 in the fluidic chip 101 described above may be a 76 mm x 26 mm glass slide (such as a CARL ROTH, 0656 glass slide).

[0163] The first material, second material and / or third material are not made of PDMS.

[0164] Preferably, each slide as described herein is a standalone element which is made of a single material ( / .e. the first, second or third material, respectively).

[0165] As another option, the first slide can be part of a multilayer element, such as a laminated element. In this case, the first slide (made of the first material) is an outermost layer of the multilayer element, which faces the second slide.

[0166] As another option, the third slide (if present) can be part of a multilayer element, such as a laminated element. In this case, the third slide (made of the third material) is an outermost layer of the multilayer element, which faces the second slide.

[0167] The second adhesive composition may be the same or different from the first adhesive composition. It is preferably the same.

[0168] Each adhesive composition may comprise a cyclic olefin copolymer (COC), an epoxy resin, an acrylate resin, a polyamide, polyethylene terephthalate, polyethylene naphthalate, ethylene vinyl alcohol, polyvinyl chloride, polyvinylidene chloride and / or liquid crystal polymers.

[0169] The epoxy resin may have any one or more of the following characteristics. The epoxy resin may comprise polar groups within the epoxy resin (e.g., hydroxyl, amino, etc.). These polar groups can generate hydrogen bonding between polymeric chains and also restrain segmental motion in the polymeric network. Such bonding and segment mobilities are central to the solubility and diffusivity of penetrants, such as oxygen, within the polymeric structure, the epoxy therefore allowing the chip 101 to approach the characteristics of a fully glass chip 101 . One example of a suitable adhesive composition is Loctite® 9492 by Henkel.

[0170] The acrylate based resin preferably comprises a methacrylate base monomer, mixed with a diluent monomer, for example another methacrylate monomer. The adhesive composition may comprise photo-initiators and / or inhibitors. The base monomer may comprise any one of bisphenol A glycidyl methacrylate (Bis-GMA), ethoxylated bisphenol A dimethacrylate (Bis-EMA), and urethane dimethacrylate (UDMA). The diluent monomer may comprise any one of triethylene glycol dimethacrylate (TEGDMA), decanediol dimethacrylate (D3MA), and 2-hydroxyethyl methacrylate (HEMA). The diluent monomer may act as a cross-linking diluent to adjust the viscosity of the adhesive composition. Preferably, the adhesive composition may comprise a nanofiller, such as, for example, SiCh nanoparticles. The use of such nanofiller can allow for further reducing the oxygen permeability of the adhesive composition. One example of a suitable acrylate based resin is Filtek™ Supreme XTE by 3M.

[0171] The polyamide may for example be polyamide 6.

[0172] The adhesive composition may be temperature-sensitive or pressuresensitive, i.e. adhesion by the adhesive composition may be effected or promoted by heating or by applying pressure to the adhesive joint. The adhesive composition may also be sensitive to light-initiated and / or a chemically initiated curing. This can allow for an accelerated curing time without need for applying heat to the chip 101 , allowing for the chip 101 to be quickly ready to use once assembled, without damaging the architecture of the chip 101 in the process. The adhesive composition may for example be UV-sensitive.

[0173] The geometric configuration of the first adhesive layer and of the second adhesive layer (if present) are preferably adjusted in order to minimize oxygen permeation to the channel and from the channel. This is especially the case when the first slide, second slide and third slide (if present) are made of glass or a similar very oxygen-impermeable material.

[0174] The first adhesive layer 114 may be evenly distributed between the first and second slides 100, 104. The second adhesive layer 116 may be evenly distributed between the second and third slides 104, 108. Each adhesive layer 114, 116 may have a thickness of 300 pm or less, preferably 200 pm or less, more preferably 150 pm or less, most preferably 100 pm or less. By way of example, the thickness may range from 20 pm to 150 pm, or from 80 pm to 120 pm. Minimizing the adhesive layer thickness is generally favorable to minimize oxygen permeation. However, it may be desirable to avoid using an excessively small adhesive layer thickness in order to keep the fluidic chip easy to manufacture.

[0175] The surface of the walls of the channel 106 delimited by one or both adhesive layers 114, 116 may for example be less than 10%, such as from 1 % to 5%, relative to of the total surface of the walls of the channel 106 intended to be in contact with liquid medium.

[0176] The first adhesive layer 114 and / or the second adhesive layer 116, if present, extends in contact with the second slide 104 over a distance (from the channel and towards the respective edges of the second slide 104, in the XY plane parallel to the second slide 104) of from 1 mm to 50 mm, preferably from 2 mm to 20 mm, more preferably from 5 mm to 15 mm (around all edges of the channel). This distance w is illustrated in FIG. 2B, which shows one adhesive layer 114 in contact with the second slide 104. This distance w also corresponds to the dimension (in the XY plane) of the adhesive layer from the channel, to the closest edge of the adhesive layer extending away from the channel. For the sake of convenience, it is designated herein as the “width" of the adhesive layer.

[0177] In some variations, this width w can be constant over the entire perimeter of the channel (in the XY plane). In other cases, this width w can vary over the perimeter of the channel (in the XY plane); then the abovementioned ranges apply to the average width (when integrated over the entire perimeter of the channel).

[0178] When the channel has a substantially elongated rectangular shape (in the XY plane), the width w may substantially correspond to the (constant or average) dimension of the adhesive layer in the XY plane, perpendicular to the longitudinal axis of the channel, from the channel to the closest edge of the adhesive layer extending away from the channel (the contribution of the width of the adhesive layer parallel to the longitudinal axis, at both ends of the channel, being generally negligible).

[0179] It has been found by the present inventors that it is desirable, for the purposes of control of dissolved oxygen concentration within the channel, if this width is not excessively small and not excessively large. An excessively large width negatively impacts the reaching of a low oxygen target value by flowing oxygen depleted liquid medium to the channel, while an excessively small width negatively impacts the maintenance of a low oxygen target value upon stopping the flow of oxygen liquid medium to the channel.

[0180] The first adhesive layer 114 and / or the second adhesive layer 116 may cover at least 70% of the surface of the second slide 104, preferably at least 80%, at least 90%, at least 95% or at least 98%. Preferably, substantially the entire surface of the second slide 104 facing the first slide 100 is covered with the first adhesive layer 114, and / or substantially the entire surface of the second slide 104 facing the glass slide 108 is covered with the second adhesive layer 116.

[0181] Method for fabricating the microfluidic chip

[0182] It is also provided a method for fabricating the fluidic chip 101. The fluidic chip 101 may comprise any one or more of the features described above.

[0183] As indicated by arrow 204 of FIG. 3, and making reference to a fluidic chip comprising a five-layer assembly, the method comprises providing the first adhesive layer 114 between the first slide 100 and the second slide 104. For example, the first slide 100 may be coated with the first adhesive composition before being joined to the second slide 104; or the second slide 104 may be coated with the first adhesive composition before being joined to the first slide 100; or both slides may be coated with the first adhesive composition and then joined.

[0184] The method comprises providing the second adhesive layer 116 between the second slide 104 and the third slide 108. For example, the third slide 108 may be coated with the second adhesive composition before being joined to the second slide 104; or the second slide 104 may be coated with the second adhesive composition before being joined to the third slide 108; or both slides may be coated with the second adhesive composition and then joined.

[0185] The method comprises placing the first slide 100, the second slide 104 and the glass 108 slide on top of one another such that the second slide 104 is between the first slide 100 and the third slide 108.

[0186] The method may comprise spreading each adhesive composition manually, for example with a pipet tip. This may facilitate a low cost fabrication of the chip 101. Alternatively, applying each adhesive composition may comprise using a dedicated system, such as, for example a spin coating machine. This may facilitate spreading an even coating (layer) of the adhesive composition to a respective slide 100, 104, 108. Bringing the slides 100, 104, 108 together may comprise pinching or pressing the slides together so as to ensure uniform spreading of the adhesive.

[0187] Pressure, heat and / or light (such as UV light) may be applied in order to effect setting of the adhesive composition.

[0188] The method may comprise waiting for the adhesive composition between two slides to harden before adding the third one. The chip 101 may be maintained at room temperature during the waiting time. The waiting time may be a duration ranging from 30 min to 5 h, for example ranging from 1 h to 4 h, for example ranging from 1 h to 3 h, for example ranging from 1 .5 h to 2.5 h, for example 2 h. Alternatively, both adhesive layers may harden at the same time after assembling all layers together.

[0189] Adding the third slide 108 may take place after the adhesive layer 114 between the first slide 100 and second slide 104 has set (or vice versa for the first slide 100 if the second slide 104 and third slide 108 are assembled first). Before assembling the layers, the method may comprise forming the channel 106 into the second slide 104. The method may also comprise forming the at least two holes 102a, 102b into the first slide 100 per channel 106.

[0190] The method may comprise forming each slide 100, 104, 108. The slides 100, 104, 108 may have the same dimensions as one another (i.e. thickness, length, width). As illustrated in FIG. 3 by arrow 200, the method may comprise using a drill to provide the channel 106 in the second slide 104. The method may comprise using a drill 120 to provide the holes 102a, 102b in the first slide 100. Use of the drill for providing such features can further facilitate the manufacturing process of the chip 101 . For example, a drill (DREMEL model 4000) at 6000 RPM may create the chamber of the central channel. Drill bits with diamond tips may be used to create two initial holes in the second glass slide 104. Each hole may have a lateral shape on the planar surface of the glass slide 104. The diamond tips may have a diameter ranging from 1 mm to 6 mm, for example 4 mm. A diamond disk may be used to create the channel 106 from the initial holes. Diamond tips may be used to create the at least two holes 102a, 102b. The tips may have a diameter ranging from 1 mm to 6 mm, for example 2 mm. The tips may form holes 102a, 102b with internal diameters ranging from 1 mm to 8 mm.

[0191] Alternatively, the channel 106 and / or holes 102a, 102b may be formed by laser ablation or by a sandblaster. This can allow for a channel 106 of more precise dimensions (for example than that formed by a drill). A laser ablation machine (C180II 12-40W CO2) can be used. The laser ablation may comprise a number of repetitions. The number of repetitions may range from 10 to 30 repetitions, for example from 15 to 25 repetitions, for example 20 repetitions.

[0192] The method may comprise plunging the slides 100, 104, 108 in water during steps of drilling or ablation. This can prevent the slides 100, 104, 108 from breaking.

[0193] As also shown by arrow 202 of FIG. 3, the chip 101 may comprise a sensor 110. The method of fabrication can be particularly suited to the inclusion of a sensor 110, as the steps of fabrication do not require use of methods that may be damaging to the sensor 110. Consequently, the sensor can remain undamaged and the quality of the performance of the sensor 110 can be maintained. The method may comprise positioning the sensor 110 on the third slide 108 (or first slide 100) before assembling the layers.

[0194] In case the fluidic chip is used for culturing a living entity (as further described below), the method may comprise adding a sample of a living entity to the channel 106. The sample can be added after making the chip, by flowing liquid medium containing the sample to the channel 106 and incubating the sample in the channel 106. Alternatively, the sample can be added to the chip during the chip manufacturing process.

[0195] The method may comprise adding the sample of a living entity to the channel 106 once the second slide 104 and third slide 108 have been connected to one another by the second layer of adhesive 116, and before connecting the first slide 100 to the second slide 104 by the first layer of adhesive 114. The addition may then be carried out by pipetting onto the surface of the third slide 108 within the channel 106. Alternatively, the method may comprise adding a sample of a living entity to the channel 106 once the second slide 104 and first slide 100 have also been connected to one another by the first layer of adhesive 114.

[0196] In particular, living entities may adhere to one or more surfaces of the channel. A coating (such as a fibronectin coating) may be provided on or more surfaces of the slides to favor adhesion of the living entities, for example cells. The cells may be pretreated, such as by putting them into contact with trypsin, prior to being added.

[0197] Alternatively, other chemical, biochemical or biological components may be added to the channel during manufacturing or after manufacturing.

[0198] As indicated by arrow 206, connectors 112 may be added to the first slide 100. The connectors 112 may be added after the setting of the first adhesive layer 114 and the second adhesive layer 116. Adding the connectors 112 to the first slide 104 may take place after the setting of the adhesive layers 114, 116 has been completed. The connectors 112 may be fixed to the first slide 104. Each connector 112 may be positioned over a respective hole 102a, 102b.

[0199] Once fabricated, the method may comprise keeping the chip for a pause duration before using it for a test or experiment. The pause duration may range from 7 h to 15 h, for example from 8 h to 14 h, for example from 8 h to 13 h, for example from 8 h to 12 h. The chip may be kept for a pause duration that is overnight. During the pause duration, the chip 101 may be maintained at an elevated temperature ranging from 55°C to 80°C, for example ranging from 60°C to 75°C, for example ranging from 65°C to 75°C, for example 70°C. Maintaining the temperature may comprise placing the chip 101 in an oven. Applying the pause duration and maintaining the chip 101 at the elevated temperature can enable the successful evaporation of any solvents in the adhesive layers 114, 116 before starting any tests or experiments. However, when a living entity or another temperature-sensitive component has been added to the channel as described above, the chip is preferably not subjected to heating.

[0200] The fluidic chip 101 may be reusable. Upon completion of a test, study or experiment, an operator may open the chip 101 and replace the sample lying in the channel 106 with a new sample. A new test, study or experiment may be executed on the new sample. Another new test, study or experiment may be executed on the new sample.

[0201] Additionally or alternatively, the fluidic chip 101 may be disposable (i.e. the chip may be intended for one use only, or for a predetermined number of uses). For example, a first chip 101 may be used to conduct one test, study or experiment comprising providing a liquid medium to a chip 101. In the case that the chip 101 is reusable, one or more other tests, studies or experiments may be executed on the same chip 101. The chip 101 may be disposed and another chip 101 may be used to conduct another test, study or experiment comprising providing a liquid medium to the chip 101. In the case that the chip 101 is reusable, one or more other tests, studies or experiments may be executed on the same chip 101. This chip 101 may too be disposed and yet another chip 101 may be used to conduct another test, study or experiment comprising providing a liquid medium to the chip 101. In the case that the chip 101 is reusable, one or more other tests, studies or experiments may be executed on the same chip 101 .

[0202] When the fluidic chip 101 is disposed of, it can optionally be recycled, as indicated by arrow 208 of FIG. 3 (for a further intended use or as part of a reversible process). Recycling may include placing the chip 101 in a bath of solvent. The solvent may be or contain ethanol or chloroform. The bath may be at a temperature ranging from 35°C to 45°C, for example 40°C. The chip 101 may remain in the bath for a cleaning duration ranging from 30 min to 2 h, for example ranging from 30 min to 1.5 h, for example for approximately 1 hour. This can allow for the removal of the fluidic connectors 112. The connectors 112 can then be recycled. The chip 101 may then be placed in an oven. The oven may be at a temperature ranging from 400°C to 600°C, for example ranging from 450°C to 550°C, for example 500°C. The chip 101 may remain in the oven for a duration ranging from 12 h to 36 h, for example ranging from 18 h to 30 h, for example for approximately 24 h. Placing the chip 101 in the oven at such a temperature and for such a duration can enable the removal of the adhesive layers 114, 116 from the slides 100, 104, 108. The slides can then be recycled.

[0203] The method may be executed in the order of firstly (arrow 200) drilling two of the first and second slides 100, 104, adding an oxygen sensor 110 to the third slide 108 (arrow 202), assembling the three slides 100, 104, 108 with the adhesive layers 114, 116 (arrow 204), adding fluidic connectors to the first slide 100 (arrow 206) and recycling (arrow 208) the chip 101 .

[0204] What has been described above in connection with a five-layer assembly can be readily adapted in case of a three-layer assembly. The manufacturing method then comprises a step of providing a second slide 104 having a reduced thickness relative to the rest of the second slide 104. The reduced thickness area forms the channel (which will be closed by the first slide 100). The second slide 104 can be made by a conventional method such as engraving or etching. The first slide 100 and second slide 104 can be bonded as described above in connection with the five-layer assembly, and all other particular or preferred features in connection with the method can be the same.

[0205] Assembly and method of using the fluidic chip

[0206] It is also provided a method for providing a liquid medium with a controlled concentration in dissolved oxygen (and preferably with a controlled flow rate) to the fluidic chip 101. Making reference to FIG. 4, the method is implemented in an assembly comprising the chip 101 and a feeding device 202 fluidically connected to it. The method is implemented by perfusing the chip 101 and in particular the at least one channel 106 described above with the liquid medium 205.

[0207] Using the fluidic chip 101 as described above to implement the method enables controlling dissolved oxygen concentration in the chip 101 (in the channel 106) in a quick and reliable manner. The method also allows to maintain the dissolved oxygen concentration at a target value or within a desired range during static periods (e.g. durations in between perfusions).

[0208] By "perfusing" is meant flowing liquid medium 205 from a feeding device 202 to the chip 101 and then preferably a collecting reservoir 214. The structure of the chip 101 allows for any processes occurring in the channel 106 in a finely controlled dissolved oxygen environment.

[0209] The feeding device 202 may be fluidically connected to the chip 101. The feeding device 202 may comprise at least one feeding module 203. The feeding device 202 and chip 101 may together form a system wherein the dissolved oxygen partial pressure within the channel is controlled with a precision of below 2 mmHg.

[0210] The feeding device 202 may be in particular configured for feeding liquid medium having a dissolved oxygen concentration (or partial pressure of oxygen) different from the dissolved oxygen concentration (or partial pressure of oxygen) of the liquid medium at equilibrium with ambient air. It may be configured for feeding liquid medium having a dissolved oxygen concentration (or partial pressure of oxygen) above the dissolved oxygen concentration (or partial pressure of oxygen) of the liquid medium at equilibrium with ambient air, and / or liquid medium having a dissolved oxygen concentration (or partial pressure of oxygen) above the dissolved oxygen concentration (or partial pressure of oxygen) of the liquid medium at equilibrium with ambient air. Preferably, the feeding device 202 is configured for feeding liquid medium having a dissolved oxygen concentration (or partial pressure of oxygen) at a target value, or within a target range, which may be adjusted or set by the user. In particular, the feeding device 202 may be configured for feeding liquid medium having a partial pressure of oxygen of less than 150 mmHg, preferably of less than 100 mmHg, preferably of less than 75 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg. This is especially useful for culturing living entities in the fluidic chip 101 in hypoxic conditions. The partial pressure of oxygen may be for example at least 5 mmHg or at least 10 mmHg.

[0211] In what is depicted in FIG. 4, a single feeding module 203 is provided. The feeding module 203 may comprise a liquid reservoir 204 containing a liquid medium 205. The feeding device 101 and liquid reservoir 204 may perfuse the chip 101 at a dissolved oxygen partial pressure ranging from 0.1 mmHg to 150 mmHg.

[0212] The liquid reservoir 204 may be provided with a gas inlet, a gas outlet, and a liquid outlet. An admission line 206 may be configured to provide a gaseous medium into the liquid reservoir 204, the admission line 206 being connected to the gas inlet of the liquid reservoir 204, and an emission line 207 may be configured to withdraw the gaseous medium from the liquid reservoir 204, the emission line 207 being connected to the gas outlet of the liquid reservoir 204.

[0213] Controlling the oxygen concentration in the chip 101 using the feeding module 202 can assist the chip in being independent from external conditions, can allow for the oxygen concentration in the chip to equal to (or substantially equal to) the pre-equilibrated oxygen concentration in the reservoir 204, and can allow for the oxygen concentration in the liquid medium 205 to remain constant.

[0214] In some embodiments, and as shown on FIG. 4, the liquid reservoir 204 may comprise an agitating element 211 such as a magnetic stirrer, and mechanical stirrer, an ultrasound or sound transducer, a vibrating element. This makes it possible to mix the gas entering the liquid reservoir 204 with the liquid present in the liquid reservoir 204 without necessarily bubbling the gas in the liquid.

[0215] In some embodiments, the chip 101 may be placed in an oven, so that it remains at a controlled temperature, for instance a constant temperature of approximately 37°C. The oven may be part of the assembly. In some other embodiments, temperatures can be controlled by a temperature control system directly integrated in the chip 101 , comprising for instance a Peltier element, or a thermalization fluid circulation, or a resistor, or a temperature sensor, or any combination thereof.

[0216] Optionally, the liquid reservoir 204 may be provided with a closable liquid inlet, in order to refill the liquid reservoir 204 or modify the composition of the liquid medium 205 if desired. The liquid reservoir 204 may be supplied with a cap 220 to seal it from the environment. The gas and liquid inlets and outlets described herein may comprise respective passageways within the cap 220. A sealing member may be provided between the cap 220 and the reservoir body, such as an O-ring, an elastomer gasket or any deformable part. The sealing member may be squeezed between the cap 220 and the reservoir body.

[0217] The feeding device 202 may comprise at least one control valve 218 associated with the admission line 206 and at least one control valve 208 associated with the emission line 207. The admission line 206 may be connected to at least one gas source 219.

[0218] According to some embodiments, the pressure at the inlet of the chip 101 (i.e. the hole 102a, 102b fluidically connected at one end to the channel 106 and fluidically connected at another end to the flow line 213 providing the liquid medium 205 to the channel 106) may be a constant inlet pressure, ranging from 400 mbar to 600 mbar, for example from 450 mbar to 550 mbar, for example 500 mbar (above atmospheric pressure). At least one of the flow lines 213, 214 may be made of a material having low oxygen permeability (as described above in connection with the multilayer structure of the fluidic chip), such as glass, so as to prevent reoxygenation in the system. At least one of the flow lines 213, 214 may comprise a glass capillary tubing. Preferably, all flow lines between the liquid reservoir 204 and the chip 101 are made of glass.

[0219] According to some embodiments, the or each gas source 219 may be a container containing a pressurized gas (or a gas composition). The or each gas source 219 may provide a controlled gas composition, a controllable pressure, a controllable flow rate, or any combination thereof. Preferably, the or each gas source 219 provides a predetermined gas composition at a pressure which may be controlled owing to a regulator on the gas source 219. Alternatively, the gas source 219 may be a gas mixer, or in other words a container comprising at least two pressurized gases (or gas compositions) separated from one another. In this case, a mixture of the separated gases can be provided into the liquid reservoir 204 via the admission line 206. It is preferable that such gas mixer is a Venturi gas mixer, or a tunable gas mixer, so that the composition of the gaseous mixture fed to the admission line 206 can be adjusted by the user.

[0220] The gas or gas composition in the gas source(s) 219 may be chosen from air, oxygen, nitrogen, carbon dioxide, carbon monoxide, nitric oxide, hydrogen sulfide or any other gases.

[0221] The method may comprise first carrying out a N2 blowing step. This allows fast oxygen removal inside the inlet reservoir and improves equilibration at low O2. This strategy is based on Fick's law, which states that the oxygen diffusion rate increases with the oxygen concentration gradient.

[0222] The liquid medium 205 may flow with a controlled concentration in oxygen and at a controlled flow rate, from the at least one liquid reservoir 204 to the chip 101 due to the pressure in the at least one liquid reservoir 204.

[0223] The term “concentration in dissolved oxygen" refers to the quantity of oxygen dissolved in a given volume of liquid at a constant temperature. According to Henry’s law, the quantity of gas which dissolves in a given volume of liquid is directly proportional to its partial pressure P in equilibrium with the liquid. The concentration in dissolved oxygen in the liquid medium 205 depends on the composition of the gaseous medium and on the pressure in the liquid reservoir 204, thus adjusting the pressure or the composition of the gaseous medium allows to control and modify (if necessary) the concentration in one or more dissolved gases in the liquid medium 205, including oxygen. The pressure, in turn, can be adjusted by controlling the valves 218, 208 of the admission and emission lines 206, 207. The composition of the gaseous medium may also be modified by independently controlling the valves 218 of the admission line(s) 206 (if there are two or more independent gas sources 219) or by controlling the gas source 219 itself.

[0224] The gas flow rate may for example be from 2 mL / min to 5 L / min. According to some preferred embodiments, the gas flow rate may be from 50 mL / min to 700 mL / min. According to other preferred embodiments, the gas flow rate may be from 2 mL / min to 50 mL / min. According to other preferred embodiments, the gas flow rate may be from 500 mL / min to 5 L / min.

[0225] By controlling the control valve(s) 218 on the admission line(s) 206 and the control valve 208 on the emission line 207, it is possible to adjust the flow rate of gaseous medium into and out of the liquid reservoir 204, and the pressure in the liquid reservoir 204.

[0226] In turn, the pressure inside the liquid reservoir 204 determines the concentration in dissolved gas(es) in the liquid medium 205 contained in the liquid reservoir 204.

[0227] The concentration in a dissolved gas is calculated based on the following equation:

[0228] [dissolved gas component] = Kh x proportion of gas component in gaseous medium x P

[0229] Kh being Henry’s constant and P being the total pressure in the liquid reservoir 204. The flow rate of the liquid medium 205 can be adjusted depending on the pressure in the liquid reservoir 204 and the counter-pressure at the liquid outlet of the chip 101 . The liquid medium 205 flow rate may be for example from 0.5 pL / min to 200 pL / min, preferably from 1 to 100 pL / min, more preferably from 2 to 50 pL / min, even more preferably from 5 to 20 pL / min (in particular for organ-on- chip applications) or 200 pL / min to 50 mL / min or 50 to 100 mL / min.

[0230] The liquid medium 205 may be provided from the liquid reservoir 204 to the chip 101 via a feeding line 213 connected to the liquid outlet of the liquid reservoir 204 and to a liquid inlet, provided by the first hole 102a, of the chip 101. A valve may be present at the liquid outlet of the liquid reservoir 204 or on the feeding line 213 in order to open or close the fluidic connection between the liquid reservoir 204 and the chip 101 (so as to be able to stop the flow of liquid medium). At an outlet of the chip 101 , liquid medium may be collected via at least one collecting line 214. The collecting line 214 may lead to at least one collecting reservoir 215.

[0231] Each of the feeding line 213 and collecting line 214 may comprise tubing, or may comprise a (e.g. molded or microfabricated) channel if it is integrated together with the reservoir 204 and / or the chip 101 within a module or cartridge.

[0232] A liquid flow sensor or flow meter 221 may be provided on the or each feeding line 213.

[0233] A liquid flow sensor or flow meter 221 may be provided on the or each collecting line 214.

[0234] According to some embodiments, the chip 101 comprises a single liquid inlet. In this case, the liquid outlet of the liquid reservoir 204 is connected to the liquid inlet of the chip 101 via the feeding line 213 in order to provide the at least one channel of the chip 101 with liquid medium having a controlled flow rate and a controlled concentration in dissolved oxygen.

[0235] According to other embodiments, the chip 101 comprises two or more liquid inlets, i.e. more than two holes 102a, 102b (not shown in FIG. 4) in the second slide 104. In this case, the liquid outlet of the liquid reservoir 204 may be connected to all liquid inlets of the chip 101 (for example in order to provide the two or more channels of the chip 101 with the liquid medium 205 having a controlled flow rate and a controlled concentration in dissolved oxygen), or each liquid inlet of the chip 101 may be connected to a different liquid outlet of the same liquid reservoir 204 (still in order to provide each channel of the chip 101 with the liquid medium 205 having a controlled flow rate and a controlled concentration in dissolved oxygen), via one or more feeding lines 213.

[0236] The chip 101 may comprise two or more liquid outlets, i.e. more than two holes 102a, 102b in the second slide 104. The liquid outlet of the chip 101 may be at ambient (atmospheric) pressure. Alternatively, the pressure at the liquid outlet of the chip 101 may be controlled owing to an outlet gas pressure source which may be the same as the outlet pressure source of the feeding device 202 or which may be different. The set pressure may be below or above atmospheric pressure. The difference between the pressure in the liquid reservoir 204 and the counterpressure at the outlet of the chip 101 may for example range from 10 mbar to 7800 mbar, and preferably from 50 mbar to 1000 mbar. In particular, the collecting reservoir 215 may be connected to a pressure controller via a gas connection.

[0237] The liquid outlet of the chip 101 may be connected to a liquid inlet of the collecting reservoir 215 via the collecting line 214. Thus, the liquid medium may exit the chip 101 and enter the collecting reservoir 215. The pressure controller makes it possible to impose a certain pressure within the collecting reservoir 215. For example, a constant pressure may be maintained in the liquid reservoir 204 and the pressure in the collecting reservoir 215 may be regulated so as to adjust the liquid flow rate.

[0238] Alternatively, the collecting reservoir 215 may comprise at least one liquid inlet, at least one gas inlet and at least one gas outlet. In this case, the configuration of the collecting reservoir 215 may be similar to the that of the liquid reservoir 204 described above (with the difference that the collecting reservoir 215 comprises at least one liquid inlet while the liquid reservoir 204 comprises at least one liquid outlet). The liquid outlet of the chip 101 may be connected to the liquid inlet of the collecting reservoir 215 via the collecting line 214. Thus, the liquid medium 205 may exit the chip 101 and enter the collecting reservoir 215.

[0239] In some variations, the method comprises a step of flowing the gaseous medium so as to reach a target dissolved oxygen concentration in the liquid medium 205, without any flow of liquid medium 205, and then a second step of flowing the liquid medium 205 having the target dissolved oxygen concentration to the chip 101. The transition from the first step to the second step may be achieved by opening a valve between the liquid reservoir 204 and the chip 101. Advantageously, the gas keeps flowing during the second step to ensure that the dissolved oxygen concentration in the liquid medium 205 remains at the targeted level (or within a desired range). In some variations, the gas flow rate in the first step is higher than the gas flow rate in the second step. Therefore, the target dissolved oxygen concentration may be relatively quickly achieved in the first step, and then the gas flow rate may be lowered to a level which is sufficient to maintain the target dissolved oxygen concentration and to reduce the gas consumption. By way of example, the gas flow rate in the first step may be from 150 mL / min to 700 mL / min, and the gas flow rate in the second step may be from 1 mL / min to 300 mL / min. In some embodiments, the method may comprise keeping the dissolved oxygen concentration in the liquid medium 205 substantially constant while varying the flow rate of liquid medium 205. This can be performed by maintaining the pressure inside the liquid reservoir 204 at a substantially constant level, and by varying the counterpressure applied at a liquid outlet.

[0240] A valve may be present at the liquid outlet 9 or on the fluidic line 26 in order to open or close the fluidic connection between the liquid reservoir 4 and the chip 101 (so as to be able to stop the flow of liquid medium).

[0241] Alternatively to the pressure controller or additionally, the assembly may comprise a flow restrictor (not illustrated). According to some embodiments, more than one flow restrictors can be comprised in the assembly. A flow restrictor may be placed upstream of, within, or downstream of the chip 101 .

[0242] According to preferred embodiments, the liquid inlet of the chip 101 may be connected to a flow restrictor. The flow restrictor (which can be for example, and not limited to, a pinch valve, or a solenoid valve, or a piezo valve, or a passive capillary, or a mechanical valve, or an electro-valve or a filter, preferably a pinch valve) can be located between the liquid outlet of the liquid reservoir 204 and the liquid inlet of the chip 101 , and particularly between the liquid flow sensor (if such sensor is present) and the liquid inlet of the chip 101 . The flow restrictor allows to modify the hydraulic resistance, therefore modifying the liquid flow rate without modifying the concentration in dissolved gas(es) in the liquid medium 205.

[0243] Although not illustrated here, the feeding device 202 may comprise at least two feeding modules 203 which are as described above. They may comprise different liquid media in their respective liquid reservoirs. Each liquid reservoir may be fluidically connected to the chip 101 as described above.

[0244] A gaseous medium or different gaseous media may flow directly to each liquid reservoir through separate admission lines dedicated to each liquid reservoir. One or more gas sources, each being equipped with a flow meter and admission valve may be connected to the admission lines of all feeding modules. An admission switching module makes it possible to orient the gaseous medium for the gas source(s) to the admission line of a feeding module. In some embodiments, the gaseous medium may flow in the admission line of only one feeding module at a time. In other embodiments, the gaseous medium may simultaneously flow in the admission lines of more than one feeding module.

[0245] The admission switching module may be a selection valve allowing to fluidically connect one inlet port to one or more outlet ports. Alternatively, each admission line may be connected to different (dedicated) gas source(s). In this case, each feeding module can operate under an even more flexible range of different flow rates, pressures and gas concentrations.

[0246] In both cases (common gas sources or dedicated gas sources for each feeding module), the gaseous medium may exit each liquid reservoir via respective emission lines. Each emission line may be provided with a dedicated control valve. Alternatively, all emission lines may be controlled by a common control valve (also referred to herein as the common emission valve). An emission switching module may be provided to open or close the fluidic connection between each emission line and the common control valve.

[0247] The pressure in each liquid reservoir may be monitored by a respective pressure sensor as described above. The gaseous and liquid media in each liquid reservoir may mix together through the use of a respective agitating element. Liquid may flow from each liquid reservoir via respective feeding lines, sequentially or in parallel, towards the fluidic chip 101 . A feeding switching module may direct the flow of liquid from one or more of the feeding lines to the fluidic chip 101 . The feeding lines may join into a single feeding line to an inlet of the fluidic chip 101 ; or the feeding lines may be independently connected to different inlets of the fluidic chip 101 .

[0248] A liquid flow sensor may be positioned between an outlet of one of the liquid reservoirs and an inlet of the culture system, e.g., between the feeding switching module and an inlet of the culture system to measure the flow rate of the liquid medium flowing into the fluidic chip 101 . After flowing through the fluidic chip 101 . , the liquid medium may be collected via at least one collecting line in a collecting reservoir as described above. According to some embodiments, the collecting reservoir may comprise a pressure controller for applying a counter pressure at the outlet of the fluidic chip 101 , as described above. An extra reservoir may be connected in parallel to the collecting reservoir, so as to bypass the fluidic chip 101 . The feeding switching module may orient the liquid medium from one or more of the reservoirs (several inlets ports) to the fluidic chip 101 and / or to the extra reservoir (two outlet ports). One possible implementation of the feeding switching module is a set of several (as illustrated: three) on / off valves connected to 3 / 2 valve.

[0249] The extra reservoir may in particular be used for any draining or purging operations. In particular, in an initial step of the method, liquid medium may be flowed to the extra reservoir in order to purge air from the system and to avoid sending air to the fluidic chip 101 . In different steps of the methods, and by acting on the various switching modules, various liquid media may be provided at different times to the fluidic chip 101 and / or to the extra reservoir.

[0250] A liquid medium may in fact, flow from any number of feeding modules 203 to the fluidic chip 101 , in sequence or in parallel.

[0251] According to some embodiments, a feeding device 203 may be fluidically connected to a plurality of fluidic chips 101 as described above. Therefore, in the method of the invention, the liquid medium 205 may flow in sequence or in parallel from the at least one liquid reservoir 204 of the feeding device 203 to multiple fluidic chips 101. Unless explicitly specified, the assembly and in particular the feeding device 203 and each fluidic chip 101 may comprise all features described above.

[0252] The liquid medium having a controlled dissolved gas concentration and a controlled flow rate may flow through a feeding line fluidly connected to a feeding manifold that supplies the liquid medium to each available fluidic chip. Optionally, the feeding manifold may be equipped with a switching module, so as to select the fluidic chip(s) to which the liquid medium flows, at each point in time. Each fluidic chip may be fluidically connected to at least one respective collecting line at an outlet thereof. Each collecting line may be fluidically connected to an inlet of a respective collecting reservoir. Pressure within the collecting reservoirs may be monitored by respective pressure sensors, and controlled by respective pressure controllers.

[0253] Alternatively, instead of being controlled by a pressure controller, the pressure in each collecting reservoir may be controlled similarly to the liquid reservoir. In other terms, each collecting reservoir may be provided with an admission line and an emission line. A control valve may be provided on the admission line and a control valve may be provided on the as emission line. The admission line may be fluidically connected to a gas source, which can be in particular the same gas source, or one of the gas sources, used for providing gaseous medium to the liquid reservoir of the feeding device. The flow rate in each collecting line may be monitored by individual liquid flow sensors.

[0254] According to some embodiments, the feeding device may comprise multiple feeding modules 203 and may be fluidically connected to multiple fluidic chips 101 . Therefore, different liquid media may be provided in a sequential and / or parallel manner to various fluidic chips. The assembly and in particular the feeding device and each fluidic chip may comprise all features described above.

[0255] The assembly may be connected to or may comprise a control unit. The control unit makes it possible for a user to control one or more than one parameter, such gas flow rate, liquid flow rate, dissolved gas concentration, pressure, etc. The control unit may comprise a graphical user interface (GUI) which allows to choose an input value for one or more than one parameter. The control unit may ensure a fully automated operation of the assembly.

[0256] The control unit may comprise one or more processors coupled to a storage medium, as well as a computer program comprising instructions stored thereon, for performing various steps described in more detail below. The control unit may receive input from any combination of the sensors mentioned above, as well as input from the user. In particular, the control unit may receive input from one or more of: a pressure sensor associated with a liquid reservoir, at least one gas flow sensor on an admission line, at least one gas flow sensor on an emission line, a liquid flow sensor and optionally a dissolved gas sensor for the liquid medium. The control unit may also receive input from any other pressure sensor, flow sensor, light sensor, pH sensor, camera, current or voltage sensor which may be present in the assembly.

[0257] The control unit may process the input data and / or the user instructions and as a result, provide instructions to the various control valves and pressure controllers, and in particular to the control valve(s) 218 on the admission line(s) 206, the control valve(s) 208 on the emission line 207 and the pressure controller connected to the emission line 207, if present. It may also provide instructions to the pressure controller connected to the collecting reservoir 215 (if present) and / or to the flow restrictor (if present).

[0258] The control unit may in particular control the aperture ratios of the various control valves. By controlling both the admission valve 218 and the emission valve 208, it is possible to adjust the flow rate of gas Q through the liquid reservoir 204, as well as the pressure P inside the liquid reservoir 204.

[0259] As a reminder, the concentration in dissolved oxygen in the liquid medium 205 varies depending on the pressure P in the liquid reservoir 204.

[0260] Besides, the flow rate of liquid medium 205 exiting the liquid reservoir 204 depends on the difference between the pressure P in the liquid reservoir 204 and the pressure Poutiiq at the outlet of the fluidic chip 101 (counter-pressure), which in some embodiments can be adjusted by the associated pressure controller.

[0261] In summary, by controlling the admission valve 218, the emission valve 208 and pressure controller downstream of the fluidic chip 101 , it is possible to independently control the concentration in oxygen dissolved in the liquid medium 205 (which depends on P) and the liquid flow rate to and through the fluidic chip 101 (which depends on P - Poutiiq). By way of example, by maintaining the pressure P in the liquid reservoir 204 constant, and by modifying the counter-pressure at the liquid outlet of the fluidic chip 101 , the flow rate of the liquid medium 205 can be varied without modifying the dissolved oxygen concentration in the liquid medium 205.

[0262] In some embodiments, the pressure controller downstream of the fluidic chip 101 can be set to a pressure which may be higher or lower than atmospheric pressure. Using a pressure lower than atmospheric pressure is useful for achieving higher liquid flow rates. Using a pressure higher than atmospheric pressure may be useful to avoid the formation of bubbles in the fluidic chip 101 .

[0263] Advantageously, a regulation algorithm is implemented in the control unit so as to adjust the control of the valves 218, 208, pressure controllers and / or flow restrictor as a function of input from the various sensors (in particular the pressure sensor, liquid flow rate sensor and gas sensor). In some preferred embodiments, said regulation algorithm involves a closed loop configuration. The regulation algorithm may be of the proportional type (P), integral type (I), derivative type (D), proportional-integral type (PI), proportional-derivative type (PD), integral- derivative type (ID) or preferably proportional-integral-derivative type (PID).

[0264] In some embodiments, the regulation algorithm may rely on fuzzy logic, artificial intelligence, deep learning or neural networks, to optimize the regulation.

[0265] In some embodiments, one or more target values are input by the user or otherwise predetermined, such as a gas flow rate and / or a liquid flow rate and / or a concentration in dissolved oxygen in the liquid medium 205, and the control unit, using the regulation algorithm, and based on inputs from one or more sensors as described above, sends instructions to the control valves, pressure controllers and / or flow restrictor in order to reach the target values as quickly as possible (or within a predetermined timeframe). As an initial phase of the regulation, the control valves 218, 208, pressure controllers and / or flow restrictor may be controlled based on preset values which may be derived from the theoretical relationships described above, or from an experimental calibration of the device, before starting the closed loop regulation per se.

[0266] By way of example, the regulation algorithm may include the following steps, in order to adjust the gas flow rate and the pressure in the liquid reservoir 204 to target values (which may be set by the user):

[0267] - Open the admission valve 218 and emission valve 208 according to a predefined table, depending on the gas flow rate setpoint.

[0268] - Read the sensed gas flow rate. - Launch a closed loop (e.g. PID) regulation on the admission valve 218 opening in order to reach and maintain the flow rate at or near the desired setpoint.

[0269] - Read the sensed pressure.

[0270] - Launch a closed loop (e.g. PID) regulation on the emission valve 208 opening in order to reach and maintain the pressure at or near the desired setpoint.

[0271] Hence, the gaseous flow rate and the pressure are regulated independently, but consecutively in the same program loop.

[0272] As another example, when more than one gas sources 219 are present, the regulation algorithm may include the following steps, in order to adjust the individual gas flow rates and the pressure in the liquid reservoir 204 to target values (which may be set by the user):

[0273] - Open the admission valve 218 and emission valve 208 according to a predefined table, depending on the setpoints.

[0274] - Read each sensed gas flow rate and launch a closed loop (e.g. PID) regulation on the corresponding admission valve 218 opening in order to reach and maintain the flow rate at or near the desired setpoint.

[0275] - Read the sensed pressure.

[0276] - Launch a closed loop (e.g. PID) regulation on the emission valve 208 opening in order to reach and maintain the pressure at or near the desired setpoint.

[0277] As a variation, the user may for instance input the total gas flow rate as well as the composition of the gas flow, and the control unit determines the setpoint individual gas flow rates required to reach these targets. For example, if three gas containers containing oxygen (or air), nitrogen and carbon dioxide are used, the user may select a target total gas flow rate, and two target gas concentrations in the liquid medium 205 (for example oxygen and nitrogen, or oxygen and carbon dioxide). The control unit then determines the individual gas flow rates required to reach these three target values, and then implements the above regulation algorithm.

[0278] In some preferred embodiments, the control unit may use information gained from the analysis of images taken by a camera, e.g., by time-lapse acquisitions. In particular, said images may provide from imaging of the fluidic chip 101.

[0279] The control unit may take information obtained from measurement data and process it to provide a certain status on the GUI. The GUI may for example, display a notification if a certain substance or combination of substances has been detected. It may equally display if a certain concentration of substances or combination of substances has been detected. It may also display concentrations measured at multiple locations, or one or more concentration differences between locations. It may also display concentrations measured at multiple points in time, or one or more concentration differences between various points in time. Depending on the substance or concentration of substance(s) measured, the control unit may implement an algorithm to translate such a measurement into a particular result other than substance or substance concentration detection. Further, the control unit may for example process the measured data to provide instructions to the various control valves and pressure controllers or to adjust the sample sizes measured by the sensors per unit of time.

[0280] In some embodiments, notably when a target partial pressure of dissolved oxygen is equal to or higher than 30 mmHg, the desired concentration in dissolved gas oxygen may be reached in two steps. More particularly, the first step may comprise the injection of a gas mixture devoid of oxygen in the liquid reservoir 204 and, once such target partial pressure is achieved, a second step may comprise the injection of oxygen, in the desired concentration. This is preferable when the concentration in dissolved oxygen is modified from a high (initial) value to a lower (final) value. Thus, this makes it possible to reach the desired (lower) concentration in the dissolved gas more rapidly.

[0281] Alternatively, notably when a target partial pressure of dissolved oxygen is lower than 30 mmHg, the desired concentration in dissolved oxygen may be reached in one step, preferably by directly injecting the dissolved oxygen in the desired concentration in the liquid reservoir 204.

[0282] Although a specific feeding device has been described in detail above, especially in relation to FIG. 4, it is understood that any other feeding device configured for feeding liquid medium having a controlled dissolved oxygen concentration can be used.

[0283] The time taken to reach a predetermined partial pressure of oxygen pC>2 from ambient oxygen pressure conditions ( / .e. response time, or the time to reach a predetermined or target partial pressure, starting from air conditions) within the channel 106 in the fluidic chip 101 may be minimal. The time taken to reach a partial pressure pCh of 15 mmHg may be less than 5 min for a chip perfusion flow rate ranging from 80 pL / min to 120 pL / min, or for example, may have a duration ranging from 30 s to 5 min for a chip perfusion flow rate ranging from 80 pL / min to 120 pL / min, or for example, a duration ranging from 30 s to 3 min for a chip perfusion flow rate ranging from 80 pL / min to 120 pL / min, or for example, a duration ranging from 30 s to 2 min for a chip perfusion flow rate ranging from 80 L / min to 120 pL / min, for example 1.5 min for a chip perfusion flow rate of 100 pL / min - the perfused liquid medium being at the target partial pressure of 15 mmHg. In comparison to a conventional method, approximately 2.5 hours are necessary to equilibrate a T75 flask with 10 mL of medium in a hypoxic incubator.

[0284] Alternatively, the time taken to reach a predetermined partial pressure of oxygen pC>2 of 15 mmHg within the channel 106 in the fluidic chip 101 from ambient oxygen pressure conditions may be less than 11 min for a chip perfusion flow rate ranging from 20 pL / min to 60 pL / min, or for example, may have a duration ranging of less than 11 min, or less than 4 min, or less than 3 min for a chip perfusion flow rate ranging from 20 pL / min to 60 pL / min, for example of 40 pL / min - the perfused liquid medium being at the target partial pressure of 15 mmHg. This time may be at least 30 seconds, generally at least 1 min.

[0285] Alternatively, the time taken to reach a predetermined partial pressure of oxygen pC>2 of 15 mmHg within the channel 106 in the fluidic chip 101 from ambient oxygen pressure conditions may be less than 40 min for a chip perfusion flow rate ranging from 5 pL / min to 15 pL / min, or for example, may have a duration of less than 40 min or less than 30 min or less than 25 min for a chip perfusion flow rate ranging from 5 pL / min to 15 pL / min, for example of 10 pL / min - the perfused liquid medium being at the target partial pressure of 15 mmHg. This time may be at least 10 min, generally at least 20 min.

[0286] The time taken to reach ambient oxygen pressure conditions within the channel 106 from a predetermined partial pressure of oxygen pC>2 of 15 mmHg (i.e. reoxygenation time, or time to back to air conditions) may be as long as possible. The time may be greater than 2 h, for example greater than 3 h, or for example greater than 4 h, or for example greater than 5 h or greater than 10 h - in static conditions, without any perfusion of fresh liquid medium in the channel. This time can be less than 200 h, such as less than 100 h.

[0287] A kit may be provided, comprising the feeding device described above, together with one or more fluidic chips as described above, the fluidic chips being intended for use with the feeding device, as an assembly as described above.

[0288] Applications

[0289] The fluidic chip, assembly (comprising the fluidic chip) and method of using the fluidic chip described above may be used for carrying out a chemical or biochemical reaction in the fluidic chip (in the channel(s) of the fluidic chip), or for culturing living entities in the fluidic chip (in the channel(s) of the fluidic chip).

[0290] A “living entity” refers to any entity showing certain attributes including responsiveness, growth, metabolism, energy transformation, aging, stem cell fate, immunology response and / or reproduction. Preferably, the term may refer to any entity capable of the processes of consumption and secretion.

[0291] The at least one living entity may in particular be a population of individual cells, including combinations of different type of cells, organoids, cellular assemblies, organelles, organs-on-chips, tissue slices, and unicellular or multicellular organisms. The cells may include, as an exemplary and non- exhaustive list, eukaryote cells, including animal cells (such as mammal cells and more specifically human cells), yeast cells, fungal cells, plant cells, protozoa; prokaryote cells, such as bacteria. Any combination of the above may also be used. Multicellular organisms may include animals, notably but not exclusively, laboratory model animals such as nematodes, embryos, notably non-human embryos (such as fish embryos), flies, eggs, plants, fungi, genetically modified organisms (GMOs). A cell culture may also be performed in order to grow viruses (i.e. , the cells may be virus-containing cells). The at least one living entity may be a sample of biological tissue, such as in particular a tumoral tissue. The cells of the at least one living entity may be arranged in 2D (i.e. monolayer) or 3D form.

[0292] The liquid medium 205 perfused to the chip 101 is preferably an aqueous solution. It may, for example, comprise at least one reagent, and / or at least one reactant, and / or at least one energy source (e.g. glucose, glutamine, acetyl-CoA), and / or a drug or other medical treatment, and / or at least one inhibitor, such as a metabolic inhibitor (e.g. mitochondrial translation inhibitor). Some examples of inhibitors are: 2-deoxy-D-glucose a.k.a. 2-DG as a glucose metabolism inhibitor, etomoxir as a fatty acid metabolism inhibitor, CB-839 as a glutamine metabolism inhibitor, a-CHCA or AZD3965 as a lactate metabolism inhibitor, trifluoromethoxy carbonylcyanide phenylhydrazone a.k.a. FCCP, carboxin, cyanide, rotenone, oligomycin and antimycin A as mitochondrial inhibitors. The liquid medium 205 is preferably a growth medium, i.e. an aqueous composition adapted to support the growth of the at least one living entity, e.g., to support the growth of a population of microorganisms or cells via the process of cell proliferation.

[0293] At least one measurement may be taken in the method so as to determine the presence or concentration in at least one substance in the liquid medium 205 in or downstream of the chip 101. Such substances may for example be any molecule considered as a nutrient and / or a metabolite. Such substances may for example include sugars (e.g. glucose, galactose, fructose), organic acids (e.g. lactate, pyruvate), salts (e.g. acetate, malate), cytokines, chemokines, amino acids (e.g. glutamine, serine, alanine, aspartate, asparagine, glycine, and / or methionine), fatty acids, phospholipids, cholesterol, acetyl-CoA. The substance may also be a virus. The substance may also be a protein or nucleic acid or a macromolecular assembly, such as extracellular vesicles, exosomes, organites, such as mitochondria.

[0294] In some embodiments, the substance is not a gas, and in particular is not a dissolved gas in the liquid medium.

[0295] In other embodiments, the substance is a dissolved gas, such as O2 or CO2, and most preferably O2.

[0296] The measurement may be taken within the chip 101. The measurement may, for example, comprise obtaining a reading from one or more sensors 110. In particular, at least one sensor 110 may be located within the chip 101 as described above (e.g. the sensor may take measurements directly within at least one channel 106 in the chip 101 ). Alternatively and / or additionally, one or more sensors may be provided upstream of the chip 101 (e.g. along a or the feeding line 213 of the chip 101 and / or at an inlet of the chip 101 ) and / or downstream of the chip 101 (e.g. along a or the collecting line 214 of the chip 101 and / or at an outlet of the chip 101 ; or in a or the collecting reservoir 215, or by taking one or more samples from a or the collecting reservoir 215 and by analyzing them separately).

[0297] In some embodiments, the method comprises an incubation step, wherein incubation is carried out within the chip 101. Then, in a subsequent measuring step, liquid medium flows through the chip 101 at a flow rate which is higher than the flow rate of the liquid medium in the incubation step (optionally, the flow rate in the incubation step may be zero). In particular, the substance to be monitored may be moved from the chip 101 to a downstream sensor 110 (as described above) owing to the flow rate in the measuring step, thus making it possible to make the measurement. The flow rate in the measuring step may also make it possible to refresh the liquid medium within the chip 101. Such incubation steps and measuring steps may alternate, e.g. in a cyclic manner.

[0298] The method may comprise flowing a liquid medium to the chip 101 wherein the oxygen concentration in the liquid medium is from 0 to 21 %, preferably from 0.1 to 15%, or from 0.2 to 10%, or from 0.5 to 5%, or from 1 to 3%. The oxygen concentration may be approximately 18.6%, for example to simulate hyperoxic conditions. The oxygen concentration may be less than 18.6%, preferably less than 5%, for example to simulate physioxic or hypoxic conditions.

[0299] The method may comprise flowing a liquid medium to the chip 101 wherein the CO2 concentration in the liquid medium is from 0 to 20%, preferably from 0.1 to 15%, or from 5 to 10%. Such CO2 concentrations may be selected to obtain a certain pH value of the liquid medium, such as for example approximately 7.4 to simulate physiological conditions, or less than 7.4, or less than 7.2, such as approximately 6.9 to simulate oncological conditions.

[0300] As a reminder, a dissolved gas (e.g., oxygen) concentration in the liquid medium, corresponds to the partial pressure of dissolved gas in the liquid medium, divided by the total pressure of all dissolved gases in the liquid medium.

[0301] In some variations, a first liquid medium which is a growth medium may be injected into a chip 101 to cultivate the living entity (preferably cells) over a period of time of e.g. 1 hour to 7 days, such as approximately 1 day, before injecting a second liquid medium different from the first liquid medium and which is a treatment medium comprising at least one drug (e.g. a chemotherapy or immunotherapy drug) into the chip 101 over a period of time of e.g. 1 minute to 1 month, preferably 1 hour to 7 days, such as approximately 2 days.

[0302] Optionally, as a third step, a third liquid medium which is a growth medium (and which can be the same as the first liquid medium) may be provided again to the chip 101.

[0303] In some variations, a sequential supply of liquid medium to the chip 101 may be used to stimulate cells using shear stress. This enables monitoring of how the stimulation may modify parameters such as, for example, their metabolism and / or their extracellular vesicles (EVs) secretion. The liquid medium may, for example, be supplied at a high flow rate to a chip 101 . Secreted components may then be collected and sent into a different chip 101 preferably in the same assembly, possibly at a different, lower flow rate. This may be useful in particular to instigate vesicle secretion in a first step and then feed the obtained vesicles to another chip 101 .

[0304] The method may be implemented so as to take measurements relating to cell secretion and / or consumption. The method according to the invention may also be implemented to perform biochemical reactions, or enzymatic reactions. In addition, according to some preferred embodiments, the method according to the invention is used for a drug screening operation, or a toxicity screening operation.

[0305] In some variations, the method of the invention may be used to test drugs promoting ROS increase. Additionally or alternatively, the method may be used to test drugs targeting metabolic properties of cells by combining metabolic inhibitors in order to increase cell death and overcome drug resistance. As the system enables sequential injection and combination of different liquid media, and determining presence or concentration of a substance, the combination of different inhibitors with chemotherapeutic drugs may be tested in a controlled environment. The provision of a plurality of feeding modules as described above makes it possible to operate under a flexible range of different flow rates, pressures and gas concentrations. This in turn allows for the living entity in the culture system to be tested under more complex conditions that may even more closely represent the desired in vivo environment (e.g. injection of a sequence of metabolic inhibitors, a sequence of treatments and / or a sequence of energy sources such as glucose, glutamine and / or acetyl-CoA).

[0306] When several fluidic chips are used in parallel, as described above, this enables the comparison of different living entity samples in real time and under exactly the same controlled conditions.

[0307] Imaging of the fluidic chip 101 may for example provide information about the number, the shape, the metabolism, the dynamics or other properties of the living entities (such as cells) in the fluidic chip 101 .

[0308] The control unit as described above may be configured to adjust a dissolved oxygen concentration in the liquid medium and / or to adjust the flow rate of the liquid medium depending on the measurements relating to the at least one substance. For example, the dissolved oxygen concentration in the liquid medium and / or the flow rate of the liquid medium may be adjusted based on the determined level of consumption of oxygen by the living entity, or based on the determined level of consumption of a nutrient (such as glucose) by the living entity, or based on the determined level of secretion of a metabolite. The adjustment may be adapted so as to maintain said level of consumption or secretion above a predetermined threshold, or below a predetermined threshold, or within a predetermined range.

[0309] EXAMPLES

[0310] Example 1 - Numerical simulations (deoxygenation)

[0311] FIG. 5 shows the results of a study carried out using finite element analysis. The analysis was carried out based on a five-layer fluidic chip 101 as described above (comprising three glass slides and two adhesive layers made of the same adhesive composition). Oxygen transport by diffusion in the adhesive layers 114, 116 of and by diffusion-convection in the liquid medium were explored.

[0312] Design parameters included the flow rate of chip perfusion (10 pL / min, 40 pL / min, 100 pL / min), the size of the channel, and the thickness of the adhesive layers (20 pm for one test and 100 pm for another test). Two adhesive materials were modelled (PDMS and COC). The oxygen diffusion coefficient value for water was Dwater = 2.5 x10-5cm2 / s. The oxygen diffusion coefficient value for PDMS was DPDMS = 3.4 x10-5cm2 / s. The oxygen diffusion coefficient value for COC was Dcoc = 4.6 x10-8cm2 / s. Oxygen concentration in the materials at the beginning of the analysis was: C02eq(water)= 0.2 mol / m3, C02eq PDMS)= 1.69 mol / m3and Co2eq COC) ~ = 1 .125 mol / m3.

[0313] A partition coefficient was used to describe Henry’s law between the adhesive material ( / .e. layers 114, 116) and the liquid : KPDMS / water= 8.45 and KCoc / water=5.625. In the adhesive layers, Fick’s law was used to describe the oxygen profile.

[0314] A fixed amount of oxygen (15 mmHg, the average oxygen concentration pC)2 for a lung tumor) was chosen as a target value and was supplied by convective flow at each of the different flow rates (10, 40, 100 pL / min). In the liquid medium, the oxygen profile was described by the diffusion-convection equation: wjth v being the speed of liquid and VC02being the gradient of concentration in O2. The speed of liquid is v =SurQfacewith Q being the flow rate (m3 / s).

[0315] The profile of the pC>2 over time during perfusion is a two-phase exponential decay. The first fast phase, depending on convection, has a characteristic time of and the second phase depending on diffusion, has a characteristic time of T2.

[0316] Each of the graphs of shows the impact of the liquid flow rate perfusing the chip 101 as a function of the response time of the system (time to reach the target partial pressure 301 of 15 mmHg).

[0317] The first graph (left) shows a curve 300 representing the impact of the liquid flow rate of 10 pL / min perfusing the chip 101 for PDMS adhesive layers 114, 116 having a thickness of 100 pm. The same graph also shows a curve 302 representing the impact of the liquid flow rate perfusing the chip 101 for COC adhesive layers 114, 116 having a thickness of 100 pm.

[0318] The second graph (center) shows a curve 304 representing the impact of the liquid flow rate of 40 pL / min perfusing the chip 101 for PDMS adhesive layers 114, 116 having a thickness of 100 pm. The same graph also shows a curve 306 representing the impact of the liquid flow rate perfusing the chip 101 for COC adhesive layers 114, 116 having a thickness of 100 pm.

[0319] The third graph (right) shows a curve 308 representing the impact of the liquid flow rate of 100 pL / min perfusing the chip 101 for PDMS adhesive layers 114, 116 having a thickness of 100 pm. The same graph also shows a curve 310 representing the impact of the liquid flow rate perfusing the chip 101 for COC adhesive layers 114, 116 having a thickness of 100 pm. Example 2 - numerical simulations (reoxygenation)

[0320] The reoxygenation process in the chip 101 was studied based on a COMSOL finite element analysis like in example 1. The working hypothesis was that flowing of a liquid medium into the channel 106 was stopped after 1 h of perfusion. The 1 h of perfusion comprised two steps. The first step comprised executing perfusion for a duration of 2 min at a flow rate of 100 pL / min. This enabled quickly reaching the target of 15 mmHg for pCh. The second step comprised executing perfusion for a duration of 58 min at a flow rate of 10 pL / min. This enabled maintaining the target.

[0321] FIG. 6 illustrates the reoxygenation process over time in channel 106. Curve 508 represents the behavior obtained using the PDMS adhesive while curve 510 represents the behavior obtained using the COC adhesive. Section 512 provides an enlarged view of the dashed box, displaying partial pressure 501 of 15 mmHg and curve 510.

[0322] Example 3 - numerical simulations (influence of thickness and width of adhesive on deoxygenation and reoxyqenation)

[0323] Still using the same approach as in examples 1 and 2, further numerical simulations were carried out to test the influence of the thickness of the adhesive layers, and of the width of the adhesive layers (as defined above), both on deoxygenation and reoxygenation.

[0324] In all of these tests, it was assumed that two adhesive layers made of COC are present, in the 5-layer configuration chip. A flow rate of 10 pL / min was assumed.

[0325] As a first part, both adhesive layers were considered as having a thickness of 100 pm, and different widths were tested, namely 0.1 mm (801 ), 1 mm (802), 10 mm (803) and 100 mm (804). The results are shown in FIG. 7A (deoxygenation on the left, reoxygenation on the right).

[0326] It was observed that the adhesive width did not have a strong influence on the slope of deoxygenation for widths of 0.1 mm, 1 mm and 10 mm. However, a width of 100 mm markedly reduces the oxygen control properties for deoxygenation slope. This is attributed to the greater amount of oxygen retained within the material in the case of an adhesive layer having an excessively large width.

[0327] As far as reoxygenation is concerned, optimal performance was achieved with the largest width of 100 mm as the distance to ambient air is very high leading to a slow re-entry of oxygen into the channel. Conversely, the least favorable results were obtained with the width of 0.1 mm. Striking a balance between reaching a short perfusion time and a very low reoxygenation can be accomplished by designing the chip with an adhesive layer width of approximately 10 mm.

[0328] As a second part, the adhesive width was set to 10 mm, and different thicknesses were tested, namely 20 pm (811 ), 100 pm (812) and 300 pm (813). The results are shown in FIG. 7B (deoxygenation on the left, reoxygenation on the right).

[0329] It was observed that the quality of oxygen control decreases with increasing thickness, both for deoxygenation and reoxygenation.

[0330] Example 4 - experiments (deoxygenation and reoxyqenation)

[0331] A five-layer fluidic chip comprising three glass slides (conventional microscopy glass slides of 76x26x1 mm, CARL ROTH, 0656). On the central glass slide, diamond tips of 3 mm diameter were used to create two holes for inserting a diamond disk to create a rectangular channel of 3mm x 37mm. Diamond tips of 3 mm diameter were used to create two holes for the top glass slide. The glass slides were immersed in water during drilling or ablation to prevent them from breaking. An oxygen optical sensor (2 mm2cut from an OXSP5-ADH Pyroscience) was added on the third glass slide (or cover slip) used as the bottom glass slide of the chip. Oxygen measurement was done with a Pyroscience board (PICO2).

[0332] The three glass slides were assembled with two layers of (the same) adhesive. The adhesive was spread manually with a pipet tip and the glass slides were pinched to spread the glue as uniformly as possible. Two adhesives were tested: an epoxy glue (Loctite® 9492) and a methacrylate-based dental resin (Filtex Suprem XTE). Both adhesives have an oxygen permeability lower than 10’9cm3(STP).cm.cm’2.s’1.cmHg’1at 25°C.

[0333] Each adhesive was allowed to dry for 2 h at room temperature before gluing the third slide. Fluidic inlets (Nanoports, IDEX N-333) were attached with an epoxy glue (Araldite® 2000). Using a micrometer, the thickness of each glass slide and the thickness of the total chip was measured in order to ensure that the thickness of the adhesive layers was approximately 100 pm.

[0334] Before starting the experiments, the chip was kept overnight in 70°C oven to ensure all the solvents are evaporated from the glue.

[0335] An oxygen controller (having a design generally corresponding to the device of FIG. 4) was used to perfuse the fluidic chip. A fused silica capillary (Postnova, Z-FSS-250350) with sleeves 1 / 16e and 1 / 32e (F-242, F-376) was used to connect the oxygen controller to the fluidic chip. A 2-step method was used to improve the deoxygenation time at low O2 by carrying out first a N2 blowing step allowing fast oxygen removal inside the inlet reservoir.

[0336] Two parameters were monitored, in order to investigate deoxygenation and reoxygenation: the perfusion time needed to reach a target of 16.5 mmHg in the channel of the fluidic chip (by flowing water having an oxygen partial pressure of 15 mm Hg); and the increase in oxygen partial pressure 2 h after cessation of the flow (after a 1 -hour perfusion with the pre-equilibrated liquid at an oxygen level of 15 mmHg).

[0337] Three different flow rates of 10, 40 and 100 pL min-1were tested.

[0338] The results are shown on FIG. 8 (deoxygenation on top, reoxygenation at the bottom). The fluidic chip comprising epoxy adhesive is designated as 403, the fluidic chip comprising the methacrylate-based dental resin is designated as 404. By way of comparison, 402 corresponds to a numerically simulated fluidic chip comprising COC adhesive layers (as discussed in the previous examples), and 401 corresponds to a numerically simulated fluidic chip made entirely of glass (without adhesive layers). On the top graph, the order of the bars is 401 , 402, 403, 404 for each of the three flow rates of 10, 40 and 100 pL min’1.

[0339] In each test, the target was achieved in under 6 min for the flow rate of 100 pL / min. In each test, the target was achieved in under 12 min for the flow rate of 40 pL / min. In each test, the target was achieved in under 45 min for the flow rate of 10 pL / min.

[0340] As far as reoxygenation is concerned, an increase of only 0.6 mmHg / h was measured with the fluidic chip comprising epoxy adhesive (403).

[0341] Example 5 - measurement of cellular respiration

[0342] Lung cancer cells A549 were cultured for 12 h in the fluidic chip described in example 4 and comprising epoxy adhesive. Cell viability was measured to be 98.8%.

[0343] As a proof of concept of the use of the fluidic chip and associated device as a cell respirometer, cycles of static-dynamic conditions were applied, using flow to refresh the chip with DMEM pre-equilibrated at a set pCh value of 50 or 80 mmHg (at a flow rate of 40 pL / min), simulating physioxia and then stopping the flow to measure cell respiration. The result is shown on FIG. 9. The A549 lung cancer cells consumed oxygen at a rate of 0.8 mmHg / min after exposure to 50 mmHg whereas they consumed oxygen at a rate of 1 .4 mmHg / min after exposure to 80 mmHg (dotted horizontal lines on the graph).

Claims

Claims1. A fluidic chip (101 ) comprising:- a first slide (100), the first slide (100) being made in a first material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;- a second slide (104) comprising a channel (106), the second slide (104) being made in a second material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;- a first adhesive layer (114) joining the first slide (100) to the second slide (104), the first adhesive layer (114) being made in a first adhesive composition having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

2. The fluidic chip (101 ) according to claim 1 , wherein the channel (106) is a through hole within the second slide (104).

3. The fluidic chip (101 ) according to claim 1 or 2, further comprising:- a third slide (108), the third slide (108) being made in a third material having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C;- a second adhesive layer (116) joining the third slide (108) to the second slide (104), the second adhesive layer (116) being made in a second adhesive composition having an oxygen permeability of 10’9cm3(STP).cm.cm’2.s’1.cmHg’1or less at 25°C.

4. The fluidic chip (101 ) according to any one of claims 1 to 3, wherein the first material is glass or fused silica; and / or the second material is glass or fused silica; and / or the third material, if present, is glass or fused silica.

5. The fluidic chip (101 ) according to any one of claims 1 to 4, wherein the first adhesive layer (114) and / or the second adhesive layer (116), if present, extends in contact with the second slide (104) over a distancefrom the channel of from 1 mm to 50 mm, preferably from 2 mm to 20 mm, more preferably from 5 mm to 15 mm.

6. An assembly, comprising:- the fluidic chip (101 ) according to any one of claims 1 to 5;- a feeding device (202) fluidically connected to the fluidic chip (101 ), the feeding device (202) configured for feeding liquid medium having a controlled concentration in dissolved oxygen to the fluidic chip (101 ).

7. The assembly of claim 6, wherein the feeding device (202) is configured for feeding liquid medium having a partial pressure of oxygen of less than 150 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg.

8. The assembly of claim 6 or 7, wherein the feeding device (202) comprises at least one feeding module (203), and each feeding module (203) comprises:- a liquid reservoir (204) containing a liquid medium (205) and provided with a gas inlet, a gas outlet and a liquid outlet,- an admission line (206) configured to provide a gaseous medium into the liquid reservoir (204), the admission line (206) being connected to the gas inlet, and- an emission line (207) configured to withdraw the gaseous medium from the liquid reservoir (204), the emission line (207) being connected to the gas outlet;- at least one control valve (218) associated with the admission line(s) (206) and at least one control valve (208) associated with the emission line(s) (207).

9. The assembly according to any one of claims 6 to 8, wherein the fluidic chip (101 ) is constructed so that the time taken to reach a predetermined oxygen partial pressure of 15 mmHg within the channel (106) from ambient oxygen pressure conditions, is less than 5 minutes, when liquid medium having an oxygen partial pressure of 15 mmHg is perfused to the fluidic chip (101 ) at a flow rate of 100 pL / min.

10. The assembly according to any one of claims 6 to 9, wherein the fluidic chip (101 ) is constructed so that the time taken to reach ambient oxygen pressure conditions from a partial pressure of oxygen of 15 mmHg within the channel (106) is greater than 2 hours, preferably greater than 10 hours, in the absence of perfusion of liquid medium within the channel (106).

11. A method of feeding liquid medium to the fluidic chip (101 ) according to any one of claims 1 to 5, comprising a step of providing liquid medium having a controlled concentration in dissolved oxygen, and a step of flowing said liquid medium having a controlled concentration in dissolved oxygen to the fluidic chip (101 ).

12. The method of claim 11 , the liquid medium having a controlled concentration in dissolved oxygen has a partial pressure of oxygen of less than 150 mmHg, preferably of less than 50 mmHg, more preferably of less than 25 mmHg.

13. The method of claim 11 or 12, comprising a step of stopping the flow of liquid medium to the fluidic chip (101 ) and retaining the liquid medium within the fluidic chip (101 ).

14. The method of any one of claims 11 to 13, which is a method for culturing a living entity in the fluidic chip (101 ).

15. A method for manufacturing the fluidic chip (101 ) according to any one of claims 1 to 14, the method comprising:- bonding the first slide (100) with the second slide (104) owing to the first adhesive layer (114);- optionally bonding the second slide (104) with the third slide (108) owing to the second adhesive layer (116), if present.

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