Microfluidic device for modeling mechanical stresses in an organ-on-a-chip

Abstract

The invention relates to a microfluidic device (10) comprising, in an overlaid manner: a plurality of upper channels (12) forming a plurality of compression chambers (18) in the same plane; an intermediate channel (14) forming an intermediate chamber (15); a lower channel (16) forming a lower chamber (17); a porous membrane (30) separating the intermediate chamber (15) from the lower chamber (17); wherein each of the compression chambers (18) is configured to vertically compress, in operation, a section of the intermediate chamber (15) and optionally a section of the lower chamber (17); each of the compression chambers (18) comprising at least one access port (20) for control thereof.

Description

Description Title: Microfluidic device for modeling mechanical stresses in an organ-on-a-chip technical field

[0001] This disclosure relates to the field of microfluidic devices, particularly for the fabrication of organs-on-a-chip. More specifically, the present invention relates to a microfluidic device adapted to reproduce the mechanical stresses that occur within an organ in vivo. The present invention also relates to a method for manufacturing such a microfluidic device. Previous technique

[0002] It is known to use microfluidic devices that faithfully reproduce the microenvironment of human organs. Such devices can be used, in particular, for testing active ingredients, for studying infectious processes, or for studying inflammatory reactions.

[0003] It has been observed that constructing devices that merely replicate the cellular and tissue microenvironment of an organ is insufficient to faithfully reproduce the organ in vivo. For example, in the case of the intestine, the intestinal epithelium exerts shear stress on the intestinal flow, and smooth muscles apply peristaltic forces that mix nutrients while propelling the flow through the intestine. These mechanical stresses affect, for instance, the interaction of cells with the active ingredient or the pathogen.

[0004] Thus, US patent application 2020 / 224136 describes a microfluidic chip containing intestinal epithelial cells co-cultured with intestinal endothelial cells, onto which flow and stretching can be applied to identify differentially expressed genes as biomarkers. This device allows for the modeling of axial stresses applied to the flow circulating within the microfluidic chip, perpendicular to the flow direction. However, such a two-dimensional model is not realistic; peristaltic forces generate a segmented flow that cannot be represented.

[0005] The paper “Pneumatically actuated microfluidic platform for reconstituting 3D vascular tissue compression” by Jungho Ahn et al. (2020) proposes a method for modeling blood vessel compression induced by biomechanical stimuli in vitro on a polydimethylsiloxane (PDMS) chip with a compression mechanism integrated with pneumatic actuation. However, this paper is limited to blood vessel compression and does not model exchanges between different channels, such as those representing an endothelial lumen and an intestinal lumen. Furthermore, this paper does not allow for the reproduction of flow segmentation.

[0006] The paper "Advanced microfluidic device designed for cyclic compression of single adherent cells" by Kenneth KY Ho et al. (2018) further describes a device comprising compression chambers capable of trapping cells and on which pressure can be applied. However, this device does not model flow with exchanges between different channels and is limited to compression motion.

[0007] Thus, the devices described above do indeed allow for the introduction of mechanical constraints into a microfluidic system. However, none of these devices can realistically model segmented movement in three dimensions. Segmented movement can occur, for example, in the intestine, during nutrient mixing. Summary

[0008] A microfluidic device is proposed, comprising, in superimposed layers: - a plurality of upper channels forming a plurality of compression chambers on the same plane; an intermediate channel forming an intermediate chamber; a lower channel forming a lower chamber; - a porous membrane separating the intermediate chamber from the lower chamber; in which each of the compression chambers is configured to vertically compress, in operation, a section of the intermediate chamber and optionally a section of the lower chamber; each of the compression chambers comprising at least one access hole for its control.

[0009] Thus, during operation, through successive compression of the compression chambers, it is possible to apply segmented vertical compression to the intermediate and, potentially, lower chambers. It is then possible to model a segmented movement in three dimensions.

[0010] Optionally, each access hole can extend parallel to the compression direction applied to the intermediate chamber section and possibly the lower chamber section.

[0011] Optionally, each of the compression chambers may include two access holes forming, in operation, a fluid inlet and outlet in each compression chamber.

[0012] Optionally, each compression chamber can form a separate unit configured to operate autonomously, without fluid transfer between two adjacent compression chambers.

[0013] Optionally, the lower and middle channels can extend one above the other along a plurality of undulations, and each upper channel can cover at least one undulation of the plurality of undulations. The undulations optimize the applied shear force and improve the morphology of the cellular tissue compared to a linear channel.

[0014] Optionally, the device can be an organ-on-a-chip. The device can be used to study the physiology and / or pathophysiology of the modeled organ, including inflammatory responses and host-microbe interactions, particularly infectious and / or non-infectious.

[0015] Optionally, the lower channel may include at least one cell type from among endothelial cells, fibroblasts, immune cells, lymphatic cells, muscle cells, neuronal cells, and microbes, for example, viruses, bacteria, archaea, fungi, and / or protists. The lower channel may model an endothelial lumen.

[0016] Optionally, the intermediate canal may include at least one cell type from among intestinal cells, immune cells, fibroblasts, and microbes. The intermediate canal can model an intestinal lumen.

[0017] Optionally, the intermediate channel and the plurality of upper channels can be separated by a layer of material. When the compression chamber is actuated, the layer of material applies vertical compression to a portion of the intermediate channel located below the actuated compression chamber.

[0018] Optionally, the thickness of the material layer separating the intermediate channel from the upper channel can be between 20 µm and 400 µm, preferably 100 µm. The intermediate channel can be compressed by the material layer between the compression chamber and the intermediate chamber.

[0019] Optionally, the width of each channel is between 200 µm and 1000 µm, preferably 750 µm. Optionally, the height of each upper channel is between 0.2 mm and 1.5 mm, preferably approximately 1 mm. Optionally, the height of the middle channel can be between 100 µm and 800 µm, preferably 400 µm, and the height of the lower channel can be between 50 µm and 400 µm, preferably 200 µm. The channels can then be visualized by confocal microscopy.

[0020] Optionally, the porous membrane may include pores with a diameter between 0.3 µm and 10 µm, preferably 8 µm. The pore dimensions are chosen to promote exchanges across the membrane.

[0021] In another respect, a method for manufacturing the microfluidic device is proposed, comprising: - the three-dimensional printing of: a first mold comprising the imprint of a plurality of upper channels, a second mold comprising the imprint of an intermediate channel, and a third mold comprising the imprint of a lower channel; - the fabrication of the upper channels, the intermediate channel, and the lower channel by soft lithography using the first, second, and third molds; and - the assembly of the lower channel, the porous membrane, the intermediate channel and the plurality of upper channels, so that the lower channel, the intermediate channel and the plurality of upper channels are superimposed and the porous membrane separates the intermediate chamber from the lower chamber.

[0022] This manufacturing method allows for the simple and flexible creation of microfluidic devices. The molds can be easily modified to iterate on the shape and dimensions of the channels. Furthermore, the process is repeatable, as the molds can be reused to manufacture a large number of devices.

[0023] Optionally, the first and second molds can include a plurality of pins, each pin adapted to form one of the device's access holes during channel fabrication. This eliminates the need to drill the device after molding.

[0024] Optionally, each of the first, second, and third molds may include visual indicators configured to verify the alignment of the lower channel, the middle channel, and the plurality of upper channels during assembly.

[0025] According to another aspect, it is proposed that the device be used for the study of intestinal physiology (homeostasis) and / or pathophysiology, including inflammatory responses and host-microbe relationships, particularly infectious and / or non-infectious. Brief description of the drawings

[0026] Other features, details, and advantages will become apparent upon reading the detailed description below and analyzing the attached drawings, on which: Fig. 1

[0027] [Fig. 1] schematically illustrates a top view of a microfluidic device according to one embodiment. Fig. 2

[0028] [Fig. 2] illustrates a view of the device of figure 1 according to a cross-sectional embodiment perpendicular to the extension of the channels, where the three channels are present. Fig. 3

[0029] [Fig. 3] illustrates a perspective view of a first mold, called upper, which can be used to produce a plurality of microfluidic devices of figure 1 according to one embodiment. Fig. 4

[0030] [Fig. 4] illustrates a perspective view, an enlargement of figure 3 according to an embodiment showing the details of the first mold enabling the realization of the plurality of upper channels for the microfluidic device of figure 1. Fig. 5

[0031] [Fig. 5] illustrates a perspective view of a second mold, called intermediate, which can be used to produce a plurality of devices of figure 1 according to one embodiment. Fig. 6

[0032] [Fig. 6] illustrates a perspective view of a third mold, called lower, which can be used to produce a plurality of devices of figure 1 according to one embodiment. Fig. 7

[0033] [Fig. 7] illustrates a flowchart showing the steps of a manufacturing method for the device in Figure 1 according to one embodiment. Description of the implementation methods

[0034] A microfluidic device 10 according to the invention is described below with reference to Figures 1 and 2. One application of the microfluidic device 10 is the realization of organs-on-a-chip. For example, the microfluidic device 10 can be used to model the intestine. Alternatively, the microfluidic device 10 could be used to model other organs, including human organs. The microfluidic device 10 can, for example, be used to model an organ of the exocrine system or an organ of the endocrine system.

[0035] The microfluidic device 10 of the present invention is capable of reproducing the mechanical stresses applied to the lumens of organs in vivo. For example, the microfluidic device 10 of the present invention can model the shear stresses and segmentation forces applied to the intestinal lumen and / or the epithelial lumen in an intestine in vivo.

[0036] The microfluidic device 10 comprises, in a superimposed arrangement, a plurality of upper channels 12, an intermediate channel 14, and a lower channel 16, given that the plurality of upper channels 12 are located on the same plane. By superimposed, it is understood that the upper channels 12 are located above the intermediate channel 14, and the intermediate channel 14 is located above the lower channel 16. In other words, the lower channel 16, the intermediate channel 14, and the upper channels 12 are stacked one on top of the other in that order. The lower channel 16, the intermediate channel 14, and the upper channels 12 are superimposed along a superposition direction Z.

[0037] The lower channel 16 forms a lower chamber 17. The lower chamber 17 is delimited by the walls of the lower channel 16. Cells can be placed on the walls of the lower channel 16, in which case the microfluidic device 10 is an organ-on-a-chip. For example, endothelial cells, fibroblasts, immune cells, lymphatic cells, muscle cells, neuronal cells, and / or microbes, such as viruses, bacteria, archaea, fungi, and / or protists, can be placed in the lower channel 16. Endothelial cells can form an endothelium in the lower channel 16. Fibroblasts can form connective tissue in the lower channel 16. Immune cells can be mobile or directly associated with the connective tissue or endothelium in the lower channel 16. Muscle cells can form an elastic membrane in the lower channel 16.In particular, the lower channel 16 can simulate an endothelial lumen or a blood vessel. During operation, a fluid, for example a gas or a liquid, can be injected into the lower channel 16 to simulate the flow occurring, for example, in the endothelial lumen or a blood vessel. blood vessel in order to evaluate the flow in the lower channel 16 and its interaction with the cells and the mechanical stresses applied to it.

[0038] The lower channel 16 typically extends along a principal extension direction X, normal to the superposition direction Z. The lower channel 16 can extend between two access holes 20, forming, in operation, a fluid inlet 26 and a fluid outlet 28. Thus, fluid can be injected and flow in an open circuit between the inlet and outlet 26, 28, allowing continuous control of the discharge. For example, the flow of fluid injected through the fluid inlet 26 can be laminar and can be perturbed in the direction of the fluid outlet 28, particularly by the shear forces and mechanical stresses applied to it.

[0039] The lower channel 16 can extend along the principal extension direction X by following a plurality of undulations 32 on either side of the principal extension direction X, in a plane perpendicular to the superposition direction Z. Viewed from above, the lower channel 16 can extend along a substantially sinusoidal curve. Each undulation 32 is half a period of the substantially sinusoidal curve. For example, in the illustrated example, the lower channel 16 extends along five undulations 32. However, the lower channel 16 could also include a number of undulations 32 less than or greater than five. The undulations 32 allow for optimization of the applied shear force and improve the morphology of the cellular tissue compared to a linear channel.

[0040] The inferior canal 16 may have a rectangular cross-section. The width I3 of the inferior canal 16 may be between 400 µm and 1000 µm, preferably 750 µm. The height h3 of the inferior canal 16 may also be between 050 µm and 400 µm, preferably 200 µm. The dimensions of the inferior canal 16 are generally chosen to allow its visualization by confocal microscopy.

[0041] The intermediate channel 14 is positioned above the lower channel 16 along the Z-axis. The intermediate channel 14 forms an intermediate chamber 15. The intermediate chamber 15 is delimited by the walls of the intermediate channel 14. Cells can be cultured on the walls of the intermediate channel 14 to simulate the microenvironment of a lumen in an organ-on-a-chip. The intermediate channel 14 can, for example, receive intestinal cells, immune cells, and / or microbes, such as viruses, bacteria, archaea, fungi, and / or protists. The intestinal cells can form an intestinal epithelium. In this case, the intermediate channel 14 simulates the intestinal lumen.

[0042] Preferably, the lower chamber 17 and the intermediate chamber 15 can be separated by a porous membrane 30 such that a lower wall of the intermediate channel 14 and an upper wall of the lower channel are formed by the porous membrane 30. Such a membrane 30 allows the exchange of nutrients, oxygen, active ingredients, or other metabolic waste products between the lower channel 16 and the intermediate channel 14. The porous membrane 30 thus makes it possible to simulate natural physiological membranes. The porous membrane 30 can, for example, have a thickness ranging from 10 µm to 200 µm. For example, the thickness can be approximately 10 µm, 16 µm, 20 µm, 50 µm, 80 µm, or 200 µm. The membrane 30 can include The pores are circular, oval, or polygonal in shape, including octagonal and hexagonal. The pores have a diameter between 0.4 µm and 10 µm, preferably 8 µm. Diameter here refers to the largest dimension between two opposite edges of a pore. The pore dimensions are chosen to facilitate exchanges across membrane 30, between the intermediate and lower channels 14, 16. Membrane 30 is, for example, a PET membrane.

[0043] The intermediate channel 14 also typically extends along the principal extension direction X. The intermediate channel 14 can extend between two access holes 20, forming, in operation, a fluid inlet 22 and a fluid outlet 24. Thus, a fluid, for example a gas or a liquid, can be injected and flow in an open circuit between the fluid inlet and outlet 22, 24, allowing continuous control of the discharge. For example, the flow of the injected fluid into the fluid inlet 22 can be laminar and can be perturbed during its flow towards the fluid outlet 24. It is possible to evaluate the fluid flow in the intermediate channel 14 and its interaction with the cells and the stresses applied to it. Furthermore, it is possible to modify various parameters in the intermediate channel 14, such as the pH.

[0044] The intermediate channel 14 can extend along the curve traced by the inferior channel 16, particularly the substantially sinusoidal curve. Thus, the intermediate channel 16 can extend along the undulations 32. Here again, the undulations 32 optimize the applied shear force and improve the morphology of the cellular tissue compared to a linear channel.

[0045] Intermediate channel 14 may have a rectangular cross-section. The width I2 of intermediate channel 14 may be between 200 µm and 1000 µm, preferably 750 µm. Furthermore, the height h2 of intermediate channel 14 may be between 100 µm and 800 µm, preferably 400 µm. These dimensions are chosen to allow visualization of intermediate channel 14 by confocal microscopy.

[0046] The upper channels 12 are located in the same plane and form compression chambers 18. The compression chambers 18 can be arranged regularly in the plane, along the principal extension direction X. In the example illustrated in Figure 1, three compression chambers 18 are arranged regularly in the plane. Each upper channel 12 can follow a portion of the curve traced by the lower channel 16 and the intermediate channel 14.

[0047] Each compression chamber 18 is isolated from neighboring compression chambers. Each compression chamber 18 thus forms a distinct unit. Each compression chamber 18 can therefore operate autonomously, without fluid transfer between two neighboring compression chambers. This allows for precise control of the pressure parameters within each compression chamber 18 and optimization of the compression application on the intermediate chamber 15 and, if necessary, the lower chamber 17.

[0048] In the illustrated example, each upper channel 12 covers approximately one undulation. For example, a first compression chamber 18 extends over the first undulation formed by the lower channel 16 and the intermediate channel 14, a second compression chamber 18 covers The third undulation formed by the lower channel 16 and the intermediate channel 14, and a third compression chamber 18, cover the fifth undulation formed by the lower channel 16 and the intermediate channel 14. In this example, the second and fourth undulations are not covered by a compression chamber 18. Generally, a compression chamber 18 can be provided covering each of the odd undulations formed by the lower channel 16 and the intermediate channel 14.

[0049] The upper channels 12 can be separated from the intermediate channel 14 by a layer of material 54. During operation, a fluid, such as a gas or a liquid, injected into one of the upper channels 12 causes the corresponding compression chamber 18 to expand, leading to an increase in the pressure applied to the walls of said upper channel 12. This, in turn, causes the layer of material 54 to deform towards the intermediate channel 14. The layer of material 54 then applies vertical compression to a portion of the intermediate channel 14 located below the compression chamber 18 into which the fluid is injected, along the direction of overlap. The intermediate chamber 15 at this point is compressed by the layer of material 54. In some cases, this deformation of the intermediate chamber 15 can also lead to the displacement of the porous membrane 30 separating the intermediate channel 14 from the lower channel 16.In this case, a portion of the lower channel 16 arranged below the compression chamber 18 into which the fluid is injected, along the superposition direction Z, is also crushed.

[0050] Here, a thickness d1, measured along the Z-axis superposition direction, of the material layer 54 separating the upper channels 12 from the intermediate channel 14 can be between 20 pm and 400 pm, preferably between 50 pm and 200 pm, preferably 100 pm. This distance d1 is chosen to allow the intermediate channel 14 to be compressed by the material layer 54 between the intermediate channel 14 and each compression chamber 18. The thickness of the material layer 54 directly influences its deformation capacity under applied pressure. Thus, the thickness d1 can be chosen to precisely match the mechanical behavior of the material layer 54 and accurately reproduce the physiological mechanical stresses in the microfluidic device 10.

[0051] Each compression chamber 18 typically includes at least one access hole 20 for its control. Each access hole 20 allows the injection of fluid, for example air, into each of the compression chambers 18. In the illustrated example, each compression chamber 18 is individually controlled by two associated access holes 20. The two access holes 20 allow pressure control within the associated compression chamber 18. During operation, fluid injection can be carried out sequentially in each compression chamber 18 along the main extension direction X. The sequential injection of fluid into adjacent compression chambers 18 allows for the application of segmented vertical compression to the intermediate channel 14 and optionally the lower channel 16.

[0052] Preferably, each compression chamber 18 is individually controlled by two access holes 20. In operation, one of the access holes serves as the inlet for the fluid, while The other ensures its evacuation. This configuration allows for open-circuit circulation within the compression chamber 18. Pressure control is thus optimized through directed fluid flow, guaranteeing homogeneous pressure distribution and improved responsiveness to pressure changes within the compression chamber 18.

[0053] Alternatively, a plurality of access holes 20 can be commonly controlled by a single injection hole. For example, the microfluidic device 10 may include a first injection hole fluidically connected to a plurality of access holes 20 of a group of first compression chambers 18 and a second injection hole connected to a group of access holes of a plurality of second compression chambers 18, the first compression chambers 18 alternating with the second compression chambers 18. In operation, every other compression chamber along the principal extension direction X can then be compressed simultaneously, so as to apply segmented compression to the intermediate channel 14, and possibly the lower channel 16. In other cases, the number of access hole groups of a plurality of compression chambers 18 may be greater than 2, for example 3, 4, 5, 6, etc.

[0054] Each access hole 20 extends along the superposition direction Z. In operation, the fluid inlet or outlet within each compression chamber 18 is carried out along the superposition direction Z. The fluid enters or exits vertically in each compression chamber 18. Thus, the fluid inlet or outlet is parallel to the vertical compression applied to the intermediate chamber 15 and possibly the lower chamber 17. This configuration allows for increased compression of the intermediate chamber 15 and possibly the lower chamber 17, particularly compared to an embodiment in which the access holes are perpendicular to the applied vertical compression.

[0055] As an example, each compression chamber 18 can reach a pressure greater than 1 bar, preferably greater than 600 mbar. When a vertical stress is applied by the compression chamber 18, the deformation of the cross-section of the intermediate channel 14 arranged vertically below the compression chamber 18 can be greater than 100%, preferably greater than 60%. If applicable, the deformation of the cross-section of the lower channel 16 arranged vertically below the compression chamber 18 can be greater than 100%, preferably greater than 60%. Such deformations are consistent with the deformations perceived by the intestinal lumen and the epithelial cells of the intestine.

[0056] As more clearly seen in Figure 2, each upper channel 12 can have a substantially rectangular cross-section. A width 11 of each upper channel 12 can be between 200 µm and 1000 µm, preferably 750 µm. A height h1 of each upper channel 12 can also be between 0.2 mm and 1.5 mm, preferably 1 mm.

[0057] Channels 12, 14, and 16 can be formed in a plate 34 from materials such as polydimethylsiloxane (PDMS), glass, silicon, or other polymers. The plate 34 can be produced by 3D printing, for example, from hydrogel. The plate 34 can also be produced by molding using 3D-printed molds into which PDMS is then poured. 3D printers can, for example, be... stereolithography (SLA) printers, fused deposition modeling (FDM), selective laser sintering (SLS), material jetting, multi-jet fusion (MJF) powder bed fusion, or any other type of 3D printer accessible to a person skilled in the art.

[0058] Preferably, the parts formed by molding from the plate 34 are formed from PDMS. In one example, at least the layer of material 54 between the upper channels 12 and the intermediate channel 16 is formed from PDMS. For example, a PDMS / crosslinking agent ratio could be 10:1, 15:1, or 20:1. A softer formulation (less crosslinking) leads to greater deformation under the same pressure in the compression chamber 18, while a stiffer formulation (more crosslinking) limits compression. Thus, the choice of formulation can be tailored to adjust the mechanical response of the membrane 30 to the pressure in the compression chamber, according to the specific requirements of the application.

[0059] The device 10 described above can form an organ-on-a-chip. In particular, when the intermediate channel 14 contains intestinal cells, and optionally the lower channel 16 contains endothelial cells, the device 10 forms an intestine-on-a-chip. By successively compressing the compression chambers 18 along the principal extension direction X between the inlets and outlets of the lower and intermediate channels 14, 16, the peristaltic forces applied to the intestinal lumen and possibly the endothelial lumen in vivo are mimicked. During operation, fluids introduced into the inlets of the lower and intermediate channels can be drawn towards the outlets by successive compression of the compression chambers 18. The fluids can then be studied at the outlet of the device 10, or during their flow, by confocal microscopy.

[0060] The cells in the organ-on-a-chip can be cell lines. Alternatively, the cells can be primary material cells, induced pluripotent stem cells, or cells derived from human or animal organoid fragments, which are broken down and loaded into the lower and intermediate channels and adhere to their walls. This allows for greater heterogeneity in cell types.

[0061] A method for manufacturing the microfluidic device 10 is described below with reference to figures 3 to 7.

[0062] In the case of fabrication by molding, the manufacturing method for device 10 includes the three-dimensional printing E100 of a first mold 36 (Figures 3 and 4), a second mold 38 (Figure 5), and a third mold 40 (Figure 6). The method further includes the fabrication E200 of the upper, intermediate, and lower channels 12, 14, 16 by soft lithography using the first, second, and third molds 36, 38, 40. The manufacturing method for device 10 further includes the assembly E300 of the lower channel 12, the porous membrane 30, the intermediate channel 14, and the plurality of upper channels 16.

[0063] The E100 three-dimensional printing of the first, second and third mold is described below.

[0064] Each of the first, second, and third molds 36, 38, 40 includes at least one cavity 42, 44, 46 for the formation of a channel. Note that the first, second, and third Molds 36, 38, and 40 here comprise a plurality of cavities, so that several microfluidic devices 10 can be manufactured from the same mold. For example, each of the first, second, and third molds can comprise between 3 and 9 cavities, so that between 3 and 9 microfluidic devices 10 can be manufactured from the same mold.

[0065] The first mold 36 includes at least one first cavity 42. The first cavity 42 may include a plurality of projections extending vertically from a base 48 of the first mold 36, each projection having the shape of an upper channel 16. The first cavity 42 may further include a plurality of first pins 50a. Each first pin 50a may provide an access hole 20. The access hole may be an access hole 20 to each upper channel 12 or an access hole for forming the fluid inlet or outlet 22, 24, 26, 28 of the lower and intermediate channels 14, 16. The pins eliminate the need to drill the device 10 after molding to form the access holes 20.

[0066] The second mold 38 includes at least one second cavity 44. The second cavity 44 is shaped like the intermediate channel 14. The second cavity 44 may include a projection extending vertically from a base of the second mold, the projection having the shape of the intermediate channel 14. In addition, the second mold 38 may include a plurality of second pins 50b. When the second mold 38 is superimposed on the first mold 36, the second pins 50a extend in line with the first pins 50a of the first mold 36. Each second pin 50b provides an access hole 20, in particular the access holes intended to form the fluid inlet and outlet of the lower channel 16.

[0067] The third mold 40 includes at least one third cavity 46. The third cavity 46 takes the form of the lower channel 16. The third cavity 46 may include a projection extending vertically from a base of the third mold, the projection having the shape of the lower channel 16.

[0068] Each mold 36, 38, 40 may include visual indicators 52. These visual indicators 52 may, for example, be raised or grooved marks formed in the base of each mold 36, 38, 40. The visual indicators allow for the alignment of the channels 12, 14, 16 during their assembly after molding. The visual indicators 52 may, for example, include lines, crosses, and circles to indicate the alignment and orientation of each channel during assembly. In one example, visual indicators are arranged around each cavity of each mold.

[0069] The frequency of the undulations 32 formed by channels 12, 14 is chosen to prevent the walls of the impressions 42, 44, 46 from sticking together between two undulations 32 during solidification by the printer's laser, particularly in stereolithography printers. For example, the distance d2 between two successive undulations, measured along the principal extension direction X, is between 1 and 2 mm, preferably 1.5 mm. The spatial period of the substantially sinusoidal curve traced by the lower and intermediate channels 12, 14 can be approximately 3 mm.

[0070] The E200 manufacturing process of the upper, intermediate, and lower channels 12, 14, 16 by soft lithography includes pouring material, specifically PDMS, into each of the first, second, and third molds 36, 38, 40. The material poured into the first mold 36 forms an inverted replica of the first mold 36, comprising the plurality of upper channels 16 and optionally access holes 20. The material poured into the second mold 38 forms an inverted replica of the second mold 38, comprising the intermediate channel 14 and optionally access holes 20. The material poured into the third mold 40 forms an inverted replica of the third mold 40, comprising the lower channel 12. Each inverted replica may further include a reproduction of the visual indicators 52.

[0071] The E200 manufacturing process for channels 12, 14, and 16 may include demolding each inverted replica part from its respective mold. When the first, second, and third molds 36, 38, and 40 comprise a plurality of cavities, the E200 manufacturing process may also include cutting the inverted replica parts from each mold to individualize the channels.

[0072] The E300 assembly of upper, intermediate, and lower channels 12, 14, and 16 can include the stacking, according to the stacking direction, of the lower channel 12, the porous membrane 30, the intermediate channel 14, and the plurality of upper channels 16. Inverted replica parts from the E200 manufacturing process can be stacked on top of each other. Channel alignment can be verified by reproducing the visual indicators on the inverted replica parts. Channels 12, 14, and 16 are correctly stacked when the visual indicator replicas are aligned.

[0073] The E300 assembly can be achieved by oxygen plasma treatment. Oxygen plasma treatment allows the inverted replica parts to adhere to each other. Furthermore, the porous membrane 30 can be assembled by oxygen plasma treatment and an APTES chemical treatment. Following assembly 300, the lower channel 16, the intermediate channel 14, and the plurality of upper channels 12 are superimposed, and the porous membrane 30 separates the intermediate chamber 15 from the lower chamber 17.

Claims

Demands

1. Microfluidic device (10) comprising superimposed - a plurality of upper channels (12) forming a plurality of compression chambers (18) on the same plane; - an intermediate channel (14) forming an intermediate chamber (15); - a lower channel (16) forming a lower chamber (17); - a porous membrane (30) separating the intermediate chamber (15) from the lower chamber (17); wherein each of the compression chambers (18) is configured to vertically compress, in operation, a section of the intermediate chamber (15) and optionally a section of the lower chamber (17); each of the compression chambers (18) comprising at least one access hole (20) for its control.

2. Microfluidic device (10) according to claim 1, wherein the lower channel (16) and the intermediate channel (14) extend one above the other following a plurality of undulations (32), and each upper channel (12) covers at least one undulation of the plurality of undulations (32).

3. Device according to any one of the preceding claims, wherein each access hole (20) extends parallel to the direction of compression applied to the section of the intermediate chamber (15) and optionally the section of the lower chamber (17).

4. Device according to any one of the preceding claims, wherein each of the compression chambers (18) includes two access holes (20) forming, in operation, an inlet and outlet of fluid in each compression chamber (18).

5. Device according to any one of the preceding claims, wherein each compression chamber (18) forms a separate unit configured to operate autonomously, without fluid transfer between two adjacent compression chambers.

6. Microfluidic device (10) according to any one of the preceding claims, wherein the intermediate channel (14) and the plurality of upper channels (16) are separated by a layer of material (54).

7. Microfluidic device (10) according to claim 6, wherein a thickness (d1) of the layer of material (54) separating the intermediate channel (14) from the upper channel (12) is between 20 pm and 400 pm, preferably 100 pm.

8. Microfluidic device (10) according to any one of the preceding claims, wherein a width (11, 12, 13) of each channel (12, 14, 16) is between 200 pm and 1000 pm, preferably 750 pm.

9. Microfluidic device (10) according to any one of the preceding claims, wherein a height (h1) of each upper channel (12) is between 0.2 mm and 1.5 mm, preferably about 1 mm.

10. Microfluidic device (10) according to any one of the preceding claims, wherein a height (h2) of the intermediate channel (14) is between 100 pm and 800 pm, preferably 400 pm, and a height (h3) of the lower channel (16) is between 50 pm and 400 pm, preferably 200 pm.

11. Microfluidic device (10) according to any one of the preceding claims, wherein the porous membrane (30) comprises pores having a diameter between 300 nm and 500 nm, preferably 400 nm.

12. Method of manufacturing the microfluidic device (10) according to any one of claims 1 to 11, comprising: - the three-dimensional printing of: a first mold (36) comprising the impression (42) of a plurality of upper channels (12), a second mold (38) comprising the impression (44) of an intermediate channel (14), and a third mold (40) comprising the impression (46) of a lower channel (16); - the fabrication of the upper channels (12), the intermediate channel (14) and the lower channel (16) by soft lithography using the first, second and third molds (36, 38, 40); and - the assembly of the lower channel (16), the porous membrane (30), the intermediate channel (14) and the plurality of upper channels (12), so that the lower channel (16), the intermediate channel (14) and the plurality of upper channels (12) are superimposed and the porous membrane (30) separates the intermediate chamber (15) from the lower chamber (17).

13. A manufacturing method according to claim 12, wherein the first mold (36) and the second mold (38) comprise a plurality of pins (50), each pin (50) being adapted to form one of the access holes of the device (10) during the manufacture of the channels (12, 14, 16).

14. A manufacturing method according to claim 12 or 13, wherein each of the first, second and third molds (36, 38, 40) includes visual indicators (52) configured to check the alignment of the lower channel (16), the intermediate channel (14) and the plurality of upper channels (12) during assembly.

15. Use of the device (10) according to any one of claims 1 to 11 for the study of intestinal physiology and / or pathophysiology, including inflammatory responses and host-microbe relationships, particularly infectious and / or non-infectious.

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