阅读说明：本技术 基于水凝胶的器官芯片微流控装置 (Organ chip micro-fluidic device based on hydrogel ) 是由 S·O·戈巴 于 2018-12-21 设计创作，主要内容包括：本发明总体上涉及一种器官芯片的微流控装置(10),其包括第一元件(11)、第二元件(16)和介于第一元件和第二元件之间的水凝胶层(14)。确定第一元件、第二元件和水凝胶层的形状和尺寸,以使水凝胶层在本文公开的使用条件下能够沿给定方向膨胀和收缩,特别是在体外模拟器官功能。本发明还涉及该微流控装置的制备方法以及该微流控装置在生物医学领域中的应用,特别是用于模拟器官的结构和功能。(The present invention generally relates to a microfluidic device (10) of an organ chip, comprising a first element (11), a second element (16) and a hydrogel layer (14) interposed between the first and second elements. The first element, the second element and the hydrogel layer are shaped and dimensioned to enable the hydrogel layer to expand and contract in a given direction under the use conditions disclosed herein, in particular to simulate organ function in vitro. The invention also relates to a preparation method of the microfluidic device and application of the microfluidic device in the biomedical field, in particular to the structure and function of a simulated organ.)

1. A microfluidic device (10) comprising:

a) a first element (11), said first element (11) comprising one or more chemical functional groups on a surface thereof, wherein said one or more chemical functional groups are comprised in a molecule covalently bound to said surface;

b) a hydrogel layer (14), said hydrogel layer (14) having a first face (7) and a second face (9) opposite each other, said hydrogel layer (14) comprising more than one chemical functional group, at least one of said chemical functional groups being effective to react with at least one chemical functional group comprised in a molecule covalently bound to the surface of said first element (11); and

c) a second element (16) for receiving the first element,

wherein the hydrogel layer (14) is interposed between the first element (11) and the second element (16) on a given axis (40), the given axis (40) being substantially perpendicular to the hydrogel layer (14), and the first element (11), the second element (16) and the hydrogel layer (14) having a shape and dimensions determined so as to delineate at least one microchannel (34) between the first element (11) and the first face (7) of the hydrogel layer (14) and at least one cavity (32) between the second element (16) and the second face (9) of the hydrogel (14), the at least one microchannel (34) and the at least one cavity (32) being arranged with respect to each other so that the given axis (40) intercepts the at least one microchannel (34) and the at least one cavity (32),

wherein at least one of said chemical functional groups comprised in a molecule and at least one of said chemical functional groups of said hydrogel layer (14) are covalently bound to each other, said molecule being covalently bound to a surface of said first element (11).

2. A microfluidic device (10) according to claim 1, wherein the surface of the second element (16) facing the hydrogel layer (14) comprises more than one chemical functional group and at least one of the chemical functional groups is covalently bonded to at least one chemical functional group of the hydrogel layer (14).

3. Micro-fluidic device (10) according to any of claims 1 to 2, wherein at least one of the first element (11) and the second element (16) comprises a recess (20), the hydrogel layer (14) being mounted in the recess (20).

4. A microfluidic device (10) according to any of claims 1 to 3, wherein the hydrogel layer (14) covers at least one groove formed on the surface of the first element (11) forming at least one microchannel (34).

5. Micro-fluidic device (10) according to claim 4, wherein the recess (20) is formed on the first element (11) and comprises a bottom (22) having the at least one groove.

6. The micro fluidic device (10) according to any of claims 1 to 5, wherein the micro fluidic device (10) comprises means (46) for generating a pressure difference between the at least one micro channel (34) and the at least one cavity (32).

7. Microfluidic device (10) according to any of claims 1 to 6, wherein the first element, the second element or both elements comprise a transparent or translucent material, e.g. Polydimethylsiloxane (PDMS).

8. Microfluidic device (10) according to any of claims 1 to 7, wherein the chemical functional groups comprised in the molecules covalently bound to at least a part of the surface of the first element (11) or the second element (16) comprise: a thiol group, the chemical functional groups comprised in the hydrogel layer (14) comprising vinyl sulfone groups.

9. The microfluidic device (10) according to any one of claims 1 to 8, wherein the modulus of elasticity of the hydrogel layer (14) is in the range of 1 to 50 kPa.

10. The microfluidic device (10) according to any one of claims 1 to 9, wherein the thickness of the hydrogel layer (14) is in the range of 30 μ ι η to 500 μ ι η.

11. The microfluidic device (10) according to any one of claims 1 to 10, wherein the device comprises two or more layers of hydrogel.

12. The microfluidic device (10) according to any one of claims 1 to 11, wherein the hydrogel layer (14) comprises a polymeric substrate comprising or consisting of one or more macromers having a hydrophilic functional group attached to a polymeric backbone of the one or more macromers, optionally the polymeric substrate comprises polyethylene glycol (PEG).

13. The microfluidic device (10) according to any one of claims 1 to 12, wherein the polymeric substrate of the one or more macromers comprises a vinyl sulfone group or a thiol group or both, the molar excess of thiol groups being 0 to 10% with respect to vinyl sulfone groups.

14. The microfluidic device according to any one of claims 1 to 13, wherein the hydrogel layer comprises a microstructure or micropattern.

15. Microfluidic device (10) according to any one of claims 1 to 14, wherein at least one cell adhesion molecule is present on the first side (7) of the hydrogel layer (14), the second side (9) of the hydrogel layer (14), the body of the hydrogel layer (14), or any combination thereof.

16. The microfluidic device according to claim 15, wherein the cell adhesion molecule, such as a cell adhesion protein, is covalently linked to at least one chemical functional group of the hydrogel layer.

17. The microfluidic device according to claim 16, wherein the cell adhesion molecule is labeled with an fc antibody fragment or modified with a heterobiofunctional protein cross-linker such as heterobifunctional NHS-PEG-maleimide linker.

18. The microfluidic device according to claims 15 to 17, wherein the cell adhesion molecule comprises any one of fibronectin, collagen, laminin, or any combination thereof.

19. The microfluidic device (10) according to any one of claims 1 to 18, wherein cells are deposited on the cell adhesion molecules of the first side (7) of the hydrogel layer (14), the second side (9) of the hydrogel layer (14), the bulk of the hydrogel layer (14), or any combination thereof.

20. Microfluidic device according to claim 19, wherein the first side (7) of the hydrogel layer (14), the second side (9) of the hydrogel layer (14), the body of the hydrogel layer (14) or any combination thereof comprises cells of the same or different type.

21. The microfluidic device according to claim 19 or 20, wherein the cells form one or more layers.

22. The microfluidic device according to any one of claims 18 to 21, wherein the cell comprises a mammalian cell, in particular a human cell.

23. Method for producing a microfluidic device (10) according to any one of claims 1 to 22, comprising the steps of:

a) producing or providing a first element (11) and a second element (16);

b) -functionalizing the surface of the first element (11), e.g. PDMS, with molecules comprising more than one chemical functional group;

c) optionally, functionalizing the surface of the second element (16) such as PDMS with molecules comprising more than one chemical functional group;

d) producing or providing a hydrogel layer (14) comprising one or more chemical functional groups, the hydrogel layer (14) being effective to react with at least one chemical functional group of molecules on the surface of the first element (11) or the second element (16) or both;

e) -placing the hydrogel layer (14) between the first element (11) and the second element (16);

f) allowing at least one chemical functional group of a molecule on the surface of the first element (11), the second element (16) or both elements to covalently react with at least one chemical functional group in the hydrogel layer (14).

24. The method according to claim 23, wherein the hydrogel layer (14) is produced by cross-linking a vinyl sulfone-functionalized polyethylene glycol (PEG-VS) macromer and a thiol-functionalized polyethylene glycol (PEG-SH) macromer.

25. Method of driving a hydrogel layer (14) of a microfluidic device (10) according to any of claims 1 to 22, comprising the steps of:

a) introducing a liquid or gas into at least one microchannel (34) between the first element (11) and the first face (7) of the hydrogel layer (14);

b) introducing a liquid or gas into at least one cavity (32) between the second element (16) and the second side (9) of the hydrogel layer (14);

c) flowing the liquid or gas through the at least one microchannel (34) between the first element (11) and the first face (7) of the hydrogel layer (14);

d) flowing the liquid or gas through the at least one cavity (32) between the second element (16) and the second side (9) of the hydrogel layer (14); and

e) adjusting or changing the flow rate of the liquid or the pressure of the gas in the at least one microchannel (34) or the at least one cavity (32) creates a pressure differential between the at least one microchannel (34) and the at least one cavity (32), expanding or contracting the hydrogel layer (14) by flexing or bending alternately in two opposite directions perpendicular to the plane of the hydrogel layer towards the at least one microchannel (34) or the at least one cavity (32).

Technical Field

The present invention generally relates to an organ-on-chip (organ-on-chip) microfluidic device. More specifically, the present invention relates to a microfluidic device comprising a first element, a second element, and a hydrogel layer interposed between the first element and the second element. The first element, the second element and the hydrogel layer are shaped and dimensioned to enable the hydrogel layer to expand and contract in a given direction under the use conditions disclosed herein, in particular to simulate organ function in vitro. The devices disclosed herein allow for the development of physiologically relevant, genetic, biochemical, cellular, tissue or organ-based assays. The invention also relates to a method for producing a microfluidic device and to the use of said microfluidic device in the biomedical field, in particular for simulating the structure and function of an organ.

Background

The development of new and powerful in vitro cell assays is of critical importance to the biomedical industry. In addition to allowing basic mechanistic studies, scale-up assays are the basis of all modern drug discovery and validation procedures.

Despite great efforts, standard assays based on 2D cell culture and plastic substrates still suffer from poor reproducibility of physiological conditions, and thus most are not effective in predicting the safety and efficacy of drugs.

The main reason for these failures is the lack of physiologically relevant models available early in drug development. Academic research is also faced with problems like scarce models, especially for the study of infectious diseases where the reservoir of many pathogens is only human. Obtaining useful insight requires real-time imaging at the cellular level, which is difficult to capture in live animals. In addition, the use of small animal models remains a significant ethical issue. Therefore, alternative methods are highly desirable. Various bioengineering methods are emerging, including microfluidics, organoid (organoid) culture and combinatorial screening, as viable solutions to narrow the gap between standard in vitro culture assays and animal studies.

In recent years, new cell-based assays have emerged with the goal of providing an effective alternative to animal detection. Most remarkably, the development of human organ chips opens up a new place for collecting human cell physiology-related data.

Patent application WO 2010/009307 discloses a microfluidic device having a central microchannel separated by one or more porous membranes (e.g. one or more porous PDMS membranes) which are physically mounted. The configuration of the microfluidic device requires an additional channel adjacent to the central microchannel in order to create a pressure differential by an indirect pneumatic mechanism between the additional microchannel and the central microchannel, in response to which the membrane expands or contracts.

Patent application WO 2015/138032 discloses a biomimetic organic (organomimetic) device having a microchannel and a membrane physically fixed to two parts of the biomimetic organic device such that the membrane is regulated by a mechanical drive system physically connected thereto.

Patent application WO 2015/138034 discloses a microfluidic device having two channels of different sizes separated by a membrane. The microfluidic device employs a complex design, by utilizing additional microchannels provided therein, and by adjusting the membrane by indirect pneumatic mechanisms and/or mechanical means.

However, despite great efforts to improve the physiological relevance of cellular assays (3D cell culture, perfusion, mechanical force), existing devices require complex configurations comprising many mechanically fixed micro-components. In addition, actuation of the device relies on mechanical stimulation by external means or indirect mechanisms. Such devices require high precision mounting/alignment of miniature and complex components. Furthermore, existing devices rely on non-physiological synthetic materials that do not reproduce (repopulate) the physiological cellular microenvironment.

Disclosure of Invention

The present invention provides an advanced organ-on-chip microfluidic device that reproduces the human organ microenvironment, especially the endothelial/epithelial interface in many mechanically active compartments or tissues, by combining microfluidic and hydrogel technologies.

Hydrogel-based devices enable the cultivation of cells on a physiologically relevant substrate (hydrogel) with adjustable mechanical properties, which expands or contracts, in particular cyclically expands or contracts, under mechanical stimulation by the pressure of a fluid (liquid or gas) directly on the substrate (hydrogel) in order to simulate physiological conditions such as the beating of the heart, respiration, the peristaltic movement of the intestine or muscle stretching. In addition, the use of hydrogels sensitive to proteolytic degradation enables the seeded cells to invade and interact tightly with the soft substrate provided. In contrast to prior methods, the present invention provides improved cellular assays that are capable of accurately biophysically and biochemically reproducing the extracellular matrix interface of a target tissue.

The present invention accordingly provides a microfluidic device which relies on the actuation of the hydrogel layer, which is capable of simulating or reproducing in vitro physical and/or physiological parameters of a biological compartment or tissue, in particular a human organ.

The present invention relates to a microfluidic device comprising:

a) a first element comprising one or more chemical functional groups on a surface thereof, wherein the one or more chemical functional groups are included in a molecule that is covalently bound to the surface;

b) a hydrogel layer having first and second sides opposite one another, the hydrogel layer comprising one or more chemical functional groups, at least one of the chemical functional groups being effective to react with at least one chemical functional group comprised in a molecule covalently bonded to the surface of the first element; and

c) a second component which is used for controlling the first component,

wherein the hydrogel layer is interposed between the first element and the second element on a given axis, the given axis being substantially perpendicular to the hydrogel layer, and the first element, the second element, and the hydrogel layer having a shape and dimensions determined to delineate and/or form at least one microchannel between the first element and the first side of the hydrogel layer, and to delineate and/or form at least one cavity between the second element and the second side of the hydrogel, the at least one microchannel and the at least one cavity being arranged relative to each other such that the given axis intercepts the at least one microchannel and the at least one cavity, and,

wherein at least one of the chemical functional groups included in the molecule and at least one of the chemical functional groups of the hydrogel layer are covalently bonded to each other, the molecule being covalently bonded to the surface of the first element.

As used herein, the term "element" refers to a part, block, body, or substrate of the microfluidic device that is more rigid relative to the hydrogel layer. Accordingly, the elastic modulus of the first or second element is higher than the elastic modulus of the hydrogel layer, and the elastic modulus of the first or second element is typically sufficiently high to enable selective stimulation of the hydrogel layer when a desired degree of pressure is applied (due to fluid introduced to the microchannels of the first element and/or the cavities of the second element). This direct stimulation mechanism does not require any additional channels adjacent to the microchannels or cavities, and therefore the production of the device is relatively simple. In a particular embodiment, the element consists of a rigid material, in particular a flat rigid material part.

As used herein, the term "surface" does not necessarily represent the entire surface or the entire outer surface of the first or second element, but encompasses a portion of the entire surface or a portion of its outer surface. In a particular embodiment, the surface is the entire surface of the element or substantially the entire surface of the element.

In a specific embodiment, the molecules covalently bound to the surface of the first element form a monolayer. A monolayer refers to a monolayer of molecules. The term "monolayer" may be used interchangeably with the terms "monolayer" or "molecular monolayer" and may be referred to as a "self-assembled monolayer". In a preferred embodiment, the molecules are of one type and therefore have the same molecular formula.

The term "chemical functional group" is used as a plural noun to denote two or more "chemical functional groups," which are commonly referred to in the art of organic chemistry as "functional groups. Chemical functional groups or simply functional groups refer to specific groups of atoms that determine chemical nature and reactivity. In a particular embodiment, the chemical functional group is within a molecule or compound and determines the chemical nature or reactivity of the molecule or compound. Different types of chemical functional groups consist of different atomic groups.

The molecules covalently bound to the surface of the first element comprise at least one functional group available for carrying out a chemical reaction.

The molecule, in its original structure (i.e., prior to covalent bonding with the first element or hydrogel layer), preferably comprises two end groups (i.e., atomic groups or chemical functional groups), each end group comprising one or more chemical functional groups. The functional group on one end of the molecule is covalently bonded to the surface of the first element and the functional group on the other end of the molecule is covalently bonded to at least one functional group of the hydrogel layer.

As used herein, the term "cavity" refers to a hollow space that is free of any material that makes up the elements and the hydrogel. In one embodiment, a cavity refers to a channel, in particular a microchannel, which extends along the surface of the hydrogel layer and is intended to allow the passage or circulation of a liquid or gas. For example, the liquid comprises cell culture medium. The two ends of the channel serve as an inlet and outlet for liquid or gas. In another embodiment, a cavity refers to a chamber or container that can hold a liquid without circulating the liquid. In particular, the cavity has a diameter (parallel to the hydrogel layer) in the range of 10mm to 40mm and a height (perpendicular to the hydrogel layer) in the range of 10 μm to 20 mm.

The term "microchannel" refers to a channel having dimensions (e.g., width and height) less than 1mm, except for length. In particular, the width (parallel to the hydrogel layer) and height (perpendicular to the hydrogel layer) of the microchannels are in the range of 10 μm to 999 μm. The length of the microchannel (parallel to the hydrogel layer) extends along the first face of the hydrogel layer and is intended to allow the passage or circulation of liquid. Said length of the microchannel is in the range between 0.1mm and 15 mm. Both ends of the microchannel serve as an inlet and an outlet for the liquid or gas.

As used herein, the term "hydrogel layer" refers to a layer that can be considered as entirely flat when viewed from either the first or second side of the hydrogel layer, e.g., a sheet of hydrogel of any shape (e.g., rectangular, circular, or square). The two faces are separated by a thickness of the hydrogel layer that is less than any other dimension of the hydrogel layer. The term "hydrogel" encompasses a polymer network of cross-linked hydrophilic polymers or macromonomers capable of swelling by absorbing or trapping water molecules within their structure. In one embodiment, the thickness of the hydrogel layer is uniform over its entire surface. In another embodiment, the thickness of the hydrogel layer is non-uniform across its surface (particularly in the area where the first side of the hydrogel faces the microchannel and the second side of the hydrogel faces the cavity). For example, the hydrogel layer may have a varying thickness or a lower thickness in the region relative to the remainder of the hydrogel layer.

The inventors advantageously designed a new organ-chip platform that relies on the actuation of the hydrogel layer. The apparatus includes a hydrogel layer chemically bonded thereto by forming covalent bonds, enabling the hydrogel layer to expand or contract in response to a fluid pressure differential between microchannels on one side (i.e., a first side) of the hydrogel layer and cavities on an opposite side (i.e., a second side) of the hydrogel layer. Technical advantages associated with such apparatus assembly include reduced complexity of mechanical operation of the components and improved long-term stability of the apparatus. In particular, the irreversible covalent attachment of the hydrogel layer contributes to the simplicity and direct actuation of the device, while reducing the risk of accidental disassembly, rupture or leakage. The design of the apparatus further ensures that no pressure is applied to the undesired driven portions of the hydrogel layer (i.e., the hydrogel layer is not squeezed between the two elements/components within the apparatus); as mentioned above, the only pressure experienced by the hydrogel layer is caused by the actuation of the hydrogel layer based on the fluid pressure differential. The present invention demonstrates for the first time how hydrogels can be used as force sensors in biological devices.

The hydrogel layer disclosed herein is capable of regenerating mesenchyme (also known as Stroma (Stroma)). The hydrogel layer is not inert; for example, it is susceptible to proteolytic degradation. The hydrogel layer also allows for a chemical gradient to be created between the microchannels and the cavities on the opposite side of the hydrogel layer.

According to the invention, the arrangement of the microfluidic device disclosed herein enables the hydrogel layer to be driven and deformed in a direction towards the at least one microchannel between the first element and the first side of the hydrogel layer or towards the at least one cavity between the second element and the second side of the hydrogel layer, upon stimulation of the hydrogel layer, for example by applying a fluid pressure difference between the at least one microchannel and the at least one cavity, the at least one microchannel and the at least one cavity being arranged relative to each other on a given axis that intercepts the at least one microchannel and the at least one cavity.

As used herein, the term "actuation of the hydrogel layer" refers to the act of inducing the hydrogel layer to move or elastically deform.

As used herein, the term "deformation of the hydrogel layer" refers to a reversible change or change in the form of the hydrogel layer, in particular by expansion or contraction of the hydrogel layer. In particular, the deformation is reversible and can be stopped by stimulating the hydrogel layer.

The behavior of expansion and contraction is characterized by a cyclic stretching of the hydrogel layer, since the pressure to which the hydrogel layer is subjected is due to the pressure exerted by the fluid perpendicular to the contact surface (first or second side) of the hydrogel, causing the hydrogel layer to bend or contract. In particular, the pressure experienced by the hydrogel layer is due to the difference in fluid pressure between the microchannels on the first side of the hydrogel and the cavities on the second side of the hydrogel.

As used herein, the term "stimulation of the hydrogel layer" refers to the application of a stimulus that drives the hydrogel to move or elastically deform (i.e., move the hydrogel) by expanding or contracting in response to a pressure differential between a microchannel delineated by the first element and a first side of the hydrogel layer and a cavity between the second element and a second side of the hydrogel layer.

The pressure difference, in particular the fluidic pressure difference, is generated by the fluid (gas or liquid) flowing in the microchannels on the first side of the hydrogel and the fluid (gas or liquid) flowing in the cavities on the second side of the hydrogel. The fluid exerts varying amounts of pressure on the first and second sides of the hydrogel layer by adjusting the flow rate of the liquid and/or the pressure of the gas.

Thus, the actuation of the hydrogel layer is actuated by varying the liquid flow rate or the gas pressure in the microchannels and/or cavities.

The fluid in the microchannels on the first side of the hydrogel layer and the fluid in the cavities on the second side of the hydrogel layer may flow in the same direction or in opposite directions.

In one embodiment, the fluid in the at least one microchannel on the first side of the hydrogel layer or in the at least one cavity on the second side of the hydrogel layer is a gas. The use of gas to apply pressure to the hydrogel layer may allow, for example, alveolar reconstruction.

In one embodiment, the fluid in the at least one microchannel on the first side of the hydrogel layer or in the at least one cavity on the second side of the hydrogel layer is a liquid. The use of a liquid allows better control of the pressure within the cavity and thus the adjustment (i.e. elastic deformation) of the hydrogel layer due to its low compressibility compared to a gas.

In a preferred embodiment, the fluid in all microchannels and chambers comprised in the device is a liquid.

In one embodiment, the first element is in physical contact with the first side of the hydrogel layer, and the second element may or may not be in contact with the second surface of the hydrogel layer.

In another embodiment, the second element is in physical contact with the first element and the hydrogel layer is in physical contact with one or both of the first element or the second element.

In all embodiments of the invention, the contact between the first and second elements does not exert any pressure on the hydrogel layer. The first and second elements are secured to each other directly or indirectly such that the hydrogel layer is sealingly received in a fluid circuit configured with means for creating a pressure differential between the at least one microchannel and the at least one cavity.

In a particular embodiment, the surface of the second element facing the hydrogel layer comprises more than one chemical functional group, and at least one of said chemical functional groups is covalently bound to at least one chemical functional group of the hydrogel layer. The one or more chemical functional groups are included in molecules that are covalently bonded to the surface of the second member. The molecule comprising one or more chemical functional groups may be the same as or different from the molecule comprising one or more chemical functional groups covalently bound to the surface of the first element. Accordingly, the one or more chemical functional groups on the surface of the second element may be the same or different than the one or more chemical functional groups on the surface of the first element.

In another embodiment of the invention, the device may be configured such that at least one of the first and second elements is made of at least two parts, a first part and a second part. For example, the second part of the first element is located between the first part of the first element and the second element. The second part of the first element is constructed as follows: such that a first face of the second part of the first element is in contact with the first part of the first element and a second face (opposite the first face) of the second part of the first element is in contact with the second element. In another similar example, the second part of the second element is located between the first part of the second element and the first element. The second part of the second element is configured as follows: a first face of the second part of the second element is in contact with the first part of the second element and a second face (opposite the first face) of the second part of the second element is in contact with the first element.

In one embodiment, the second component of the first or second element serves as an insulating element that creates one or more cavities between the second side of the hydrogel layer and the second element. In another embodiment, the second part of the first or second element is a ring-shaped element having one or more hollow structures (e.g., hollow circles, squares, or rectangles). For example, the second part is an annular sealing element of any shape, for example circular, square or rectangular, in particular a ring with a circular cross-section, i.e. an O-ring, which prevents leakage of liquid or gas between the second side of the hydrogel layer and the second element. In a particular embodiment, the second part of the first element or the second element has a dual function: it acts as a spacer forming one or more cavities between the second side of the hydrogel layer and the second member, and as an annular sealing member preventing leakage of liquid or gas between the second side of the hydrogel layer and the second member.

In one embodiment, the first element or the second element or both comprise at least one depression or indentation therein, in particular in at least one flat side or surface thereof. The at least one depression or indentation is created by a variety of techniques including, but not limited to, photolithography, laser ablation, micro-milling, etching, or molding, and may have any shape or size. In a particular embodiment, the depression or indentation comprises at least one wall perpendicular to the surface comprising the depression or indentation, the wall defining the height or depth of the depression or indentation.

The at least one depression or indentation included in the first and second elements may have the same or different shape and size. In one embodiment, the at least one depression or indentation included in the second element has a wider width than a width of the at least one depression or indentation included in the first element, the second element forming/delineating the at least one cavity between the second side of the hydrogel layer and the second element, the first element forming/delineating the at least one microchannel between the first side of the hydrogel layer and the first element.

The term "depression" or "indentation" encompasses a groove. In a particular embodiment, the first element comprises at least one recess covered by a hydrogel layer, thereby forming at least one microchannel.

As used herein, the term "groove" refers to a narrow depression or indentation in the surface, ranging from nanometers to centimeters, particularly micrometers, created by various techniques, including but not limited to photolithography, laser ablation, micro-milling, etching, or molding. The groove may be of any shape along its extension and may have any shape in cross-section. For example, the grooves may be curved, but are preferably straight, i.e. straight, and may have a curved or rectangular cross-section.

In a specific embodiment, one side of the first element comprises a recess, which is covered by a hydrogel layer, forming a microchannel. The surface area of the first side of the hydrogel layer is dimensioned to cover the recess comprised in said side of the first element. The first face of the hydrogel layer is covalently bonded to the side of the first element comprising the groove on two surfaces separated by the width of the groove. The surface area of the two surfaces separated by the width of the groove is sufficient for the covalently bound hydrogel layer to remain attached during use of the apparatus (i.e., driven by fluid pressure differential).

According to one embodiment, at least one of the first and second elements comprises a recess (recess) in which the hydrogel layer is mounted. The hydrogel layer may cover at least one groove formed on the surface of the first element, thereby forming at least one microchannel. Also, the recess may be formed on the first element and may include a bottom having the at least one groove.

In a particular embodiment, the recess opens onto an annular surface applied onto said second element to form at least one cavity, which is arranged opposite said at least one microchannel with respect to the hydrogel layer.

The first and second elements each comprise a material or any combination of materials selected from, but not limited to: silicone rubber (i.e., polysiloxane), crystalline silicon, poly (dimethylsiloxane) (PDMS), silica (e.g., quartz and glass), thermoplastics (e.g., Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), poly (ethylene glycol) diacrylate (PEGDA), Polyurethane (PU), perfluorinated compounds (e.g., perfluoroalkoxy (Teflon PFA) and fluorinated ethylene propylene (Teflon FEP)) and polyolefins (e.g., Cyclic Olefin Copolymer (COC), cyclic olefin polymer(s), (PDMS), and (co) polymersCOP), Cyclic Block Copolymers (CBC) and polyvinyl chloride (PVC)), Polyimides (PI), polylactic-glycolic acid (PLGA), Thermosetting Polyesters (TPE), non-stoichiometric thiol-ene (OSTE), transparent ceramics (e.g. alumina (Al)₂O₃) Spinel (MgAl)₂O₄) Yttrium Aluminum Garnet (YAG) and neodymium-doped yttrium aluminum garnet (Nd: YAG)) and paper (e.g., transparent or translucent paper). In one embodiment, the first and second members comprise silica (e.g., quartz and glass). In another embodiment, the first and second elements comprise Polydimethylsiloxane (PDMS), thermoplastics (e.g., Polymethylmethacrylate (PMMA), Polycarbonate (PC), Polystyrene (PS), Polyurethane (PU), perfluorinated compounds (e.g., perfluoroalkoxy (Teflon PFA) and fluorinated ethylene propylene (Teflon FEP)) and polyolefins (e.g., Cyclic Olefin Copolymer (COC), Cyclic Olefin Polymer (COP), Cyclic Block Copolymer (CBC) and polyvinyl chloride (PVC)), polylactic-glycolic acid (PLGA), Thermoset Polyesters (TPE).

The first and second elements may or may not comprise the same material. In a particular embodiment, the first and second elements are made of the same material. In another embodiment, the first and second members are made of different materials.

In a particular embodiment, the first element and/or the second element are made of a material suitable for being able to detect a signal generated when using the microfluidic device, the signal being generated for example by an optical microscope, a fluorescence microscope or an electron microscope. The material in particular enables the detection of at least one electromagnetic wavelength emitted by the at least one microchannel and/or the at least one cavity and/or the first or second side of the hydrogel layer or the component (e.g. biological, chemical or biochemical compound) in the hydrogel body. In particular, the detection is of fluorescence.

In a preferred embodiment, at least one or all of the elements of the first, second or third elements comprise a transparent or translucent material selected from the materials listed above.

In another preferred embodiment, the first member comprises or consists of PDMS. In another preferred embodiment, the second member comprises or consists of silica.

In a particular embodiment, the chemical functional group included in the molecule covalently bonded to the surface of the first or second member comprises a thiol group (-SH). The molecule covalently bound to the surface of the first or second member may have the general formula X (CH)₂) n SiY3 comprising reactive functional groups X and Y. X is a functional group, such as a thiol group, exposed on the surface of the first element, the second element, or both, that is capable of undergoing a chemical reaction, particularly with a functional group of the hydrogel layer (e.g., a vinyl sulfone group) to form a covalent bond; n is an integer of 1 to 3; y is a functional group such as methoxy, ethoxy, methyl and the like. Y is a group covalently bonded to the surface of the first member or the second member. Examples of such molecules include, but are not limited to, (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) triethoxysilane, and (3-mercaptopropyl) methyldimethoxysilane. In a specific embodiment, the molecule comprises or consists of (3-mercaptopropyl) trimethoxysilane (MPS).

In a particular embodiment, the chemical functional group comprised in the molecule covalently bound to at least a portion of the surface of the first or second element comprises a thiol group, and the chemical functional group comprised in the hydrogel layer comprises a vinyl sulfone group. In another embodiment, the chemical functional group comprised in the molecule covalently bound to at least a portion of the surface of the first element or the second element comprises a thiol group, and the chemical functional group comprised in the hydrogel layer comprises an acrylate or maleimide group or any thiol-reactive functional group.

In a specific embodiment, the hydrogel layer has an elastic modulus (shear modulus) of 1kPa to 50 kPa. In a preferred embodiment, the hydrogel layer has a shear modulus of 10kPa to 30 kPa. The choice of the value of the elastic modulus (shear modulus) depends on the width of the microchannel or cavity, the density and thickness of the hydrogel layer, the desired degree of deformation, the presence of cells in or above the hydrogel layer (the choice of the hydrogel stiffness depends on the cells being cultured) and the temperature of the device during use. When used, a shear modulus of elasticity in the above range advantageously enables the hydrogel layer to stretch (i.e., expand or contract) by up to 25%. The degree of stretching of the hydrogel layer can be quantified by: i) comparing the area of cells seeded on the hydrogel layer before and after application of hydraulic pressure, or ii) embedding fluorescent microspheres (e.g. polystyrene magnetic beads) in the hydrogel and measuring the change in distance between the beads by means of a Traction Force Microscope (TFM).

The term "elastic modulus" refers to a numerical constant that describes the ratio of the force applied to a given area of a material, such as a hydrogel, to the deformation of the material due to the applied force. The value of elastic modulus disclosed herein refers to the shear modulus or stiffness modulus, which describes the stiffness of the material and is defined as the ratio of shear stress to shear strain. The elastic modulus (shear modulus) of the hydrogel layer is measured by techniques known in the art, such as a micro rheometer (microrheometer). For example, methods using a micro-rheometer are described in "Substrate elasticity models of the responsive of the sensory cells to the compliance documents" (Gobaa S, Hoehnel S, Lutolf MP., Integr Biol (Camb). 10.2015; 7(10):1135-42) (see, e.g., the "Measurements of substrates" paragraph). The person skilled in the art is able to determine a parameter suitable for measuring the modulus of elasticity.

In a particular embodiment, the hydrogel layer has a thickness of from 30 μm to 500 μm, more preferably from 150 μm to 350 μm, in particular from 170 μm to 340 μm, more in particular from 150 μm to 200 μm.

In a specific embodiment, the device comprises two or more layers of hydrogel. For example, the apparatus may comprise 1 to 3 layers of hydrogel, each hydrogel layer having a thickness of 30 μm to 500 μm. In one embodiment, the total thickness of the one or more hydrogel layers is in the range of 170 μm to 340 μm. In another embodiment, the thickness of one or more hydrogel layers is 170 μm. The two or more layers of hydrogel are advantageously joined together by chemical bonds (i.e., covalent bonds).

The hydrogel layer comprises a polymeric substrate comprising or consisting of a polymer or polymeric network of one or more macromers having hydrophilic functional groups attached to the polymer backbone or having hydrophilicity. In a particular embodiment, the polymeric substrate comprises polyethylene glycol (PEG), in particular functionalized PEG. In a preferred embodiment, the polymeric substrate comprises a polymeric network of two different functionalized PEG macromers (i.e., one PEG macromonomer contains one functional group and the other PEG macromonomer contains the other functional group).

In a specific embodiment, the PEG hydrogel concentration is in the range of 2.5% to 10% (w/v). In a preferred embodiment, the PEG hydrogel concentration is 5% to 10% (w/v). In another preferred embodiment, the PEG hydrogel concentration is 5% (w/v).

In a preferred embodiment, the polymeric substrate of the one or more macromers comprises a vinyl sulfone group, a thiol group, or both. The polymeric substrate of the hydrogel layer is preferably formed by crosslinking a PEG-SH macromer (in particular a star-or multi-armed PEG-SH macromer) and a PEG-VS macromer (in particular a star-or multi-armed PEG-VS macromer) by a reaction between SH groups and VS groups (in particular a Michael-type addition reaction). In this case, the covalent bond between the SH group and the VS group crosslinks the macromer to form the polymeric substrate. In a particular embodiment, the polymeric substrate comprises unsaturated SH groups and/or unsaturated VS groups.

In another embodiment, the polymeric substrate comprising PEG comprises a polypeptide as a cross-linker between PEG macromonomers, for example by using the methods disclosed in Lutolf and Hubbel (2003). The polymeric substrate comprising PEG macromers crosslinked via polypeptides is sensitive to various proteases, including metalloproteinases (MMPs).

The term "polymeric substrate" refers to a polymer network or polymeric network formed by crosslinking of macromers. In accordance with the present disclosure, macromers comprising two or more different functional groups are crosslinked to form the polymeric substrate by forming covalent bonds between the different functional groups that are susceptible to reacting with each other. Assuming that the stoichiometric molar ratio (i.e., equimolar amounts) of the two reactive functional groups is satisfied, the functional groups remain unsaturated (i.e., available for reaction) until crosslinking is complete. In a particular embodiment, the stoichiometric molar ratio of the two reactive functional groups is not satisfied, resulting in a molar excess of the unsaturated functional groups added to the polymer substrate. The functional groups serve as cross-linking linkages in the formation of the polymeric substrate.

The term "macromer" refers to a macromolecule, such as a polymer or oligomer, which acts as a precursor for subsequent crosslinking, resulting in the formation of a polymer or macromolecule having a higher molecular weight. The macromers can have respective structures/architectures including linear, cyclic, and branched structures/architectures (e.g., star, comb or brush, hyperbranched, dendritic, H-shaped, long chain branched, dumbbell-shaped, etc.).

In a specific embodiment, the hydrogel layer comprises a molar excess of thiol groups in the range of 0 to 10% relative to vinyl sulfone groups. In this case, there are unsaturated thiol groups on the bulk and surface of the hydrogel layer, which are susceptible to chemical reactions and are particularly suitable for binding to cell adhesion molecules, particularly cell adhesion molecules coupled to cross-linkers (which are covalently bound to unsaturated thiol groups).

In a specific embodiment, the hydrogel layer contains 1.2mM free thiol groups to further immobilize proteins or peptides.

In a particular embodiment, the hydrogel layer comprises a microstructure or micropattern. A topographically structured stamp (e.g., a stamp made of PDMS, silicone, glass, plastic, ceramic, or metal) produced by photolithography, micromachining, or molding may be used to form microstructures or micropatterns on at least a portion of the surface of the hydrogel layer to reproduce the structure of the target organ, particularly the crypts/villi of the intestinal tract. These stamps can be used in two ways:

1. the surface of the partially crosslinked hydrogel is subjected to soft embossing (Kobel et al, 2009). In short, the technique requires that the structured stamp be pressed onto a partially crosslinked hydrogel. After the crosslinking reaction is complete, the negative of the compressed structure is permanently transferred to the hydrogel.

2. The structure is molded while the hydrogel layer is cast (Lutolf et al, 2009). This technique requires that the hydrogel precursor solution be sandwiched between a stamp and a flat Teflon or silanized glass slide. The thickness of the spacers used determines the thickness of the structured hydrogel layer produced.

The microfluidic device of the present invention is suitable and intended for the seeding of cells, in particular mammalian cells, to perform assays including structural or functional assays related to the observation of the growth, expansion, differentiation or biological activity of cells or synthetic tissues. Accordingly, the microfluidic device allows for the seeded cells to be maintained on and/or within the hydrogel layer by adhesion molecules, such as adhesion molecules. The use of hydrogels sensitive to proteolytic degradation enables the seeded cells to invade and interact tightly with the soft substrate provided.

In a specific embodiment, the at least one cell adhesion molecule is attached (e.g., covalently or non-covalently attached) to at least one of the first side of the hydrogel layer, the second side of the hydrogel layer, the body of the hydrogel layer, or any combination thereof. The term "body of the hydrogel layer" refers to the portion between the first and second sides of the hydrogel layer that is within the thickness of the hydrogel layer. Attaching the at least one cell adhesion molecule to the hydrogel layer by: i) introduced into the body of the hydrogel layer during the step of polymerizing the hydrogel layer (e.g., may be added to a mixture of PEG macromers), and/or ii) introducing a liquid comprising cell adhesion molecules into the microchannel or cavity.

Preferably, a cell adhesion molecule, such as a cell adhesion protein, is covalently linked to the at least one chemical functional group of the hydrogel layer. In a specific embodiment, the cell adhesion molecule is covalently attached to the thiol group of the hydrogel layer.

In a particular embodiment, the cell adhesion molecule is labeled or modified to improve its adhesion function to cells. In a specific embodiment, they are labeled with fc antibody fragments or modified with heterobifunctional protein cross-linkers such as heterobifunctional NHS-PEG-maleimide linker (linker). The linker first reacts with any molecule of interest (amino acid, peptide, protein) with a primary or secondary amine in the structure at physiological pH. This will allow the formation of an amide bond between the molecule of interest and the heterofunctional linker. Thus, by adjusting the stoichiometric ratio between the target molecule and the heterofunctional linker, we can generate molecules of interest with variable maleimide functionality (typically between 1 and 20).

In a specific embodiment, the cell adhesion molecule modified with the heterobifunctional protein crosslinker is linked to the thiol group of the hydrogel layer by forming a covalent bond between said heterobifunctional protein crosslinker and said thiol group of the hydrogel layer.

In another embodiment, the cell adhesion molecule modified with the Fc antibody fragment is adsorbed onto the hydrogel layer (affinity based) containing protein a or protein G or any other molecule with affinity for the Fc fragment. Similar to the previous embodiments, protein a is covalently bound to a heterobifunctional protein cross-linker, which is covalently linked to the thiol groups of the hydrogel layer. In certain embodiments, the fluid being tested provided in the microchannel or cavity does not include an antibody or Fc fragment of an antibody.

In a particular embodiment, the cell adhesion molecule comprises any one of fibronectin, collagen, laminin, or any combination or portion thereof. In a particular embodiment, the cells are deposited on a first side of the hydrogel layer, the surface of which is previously functionalized with cell adhesion molecules, in particular cell adhesion proteins or peptides. The same operation can be performed on any of the second sides of the hydrogel layer, in the body of the hydrogel layer, or any combination thereof. In a particular embodiment, any one of, or any combination of, the first side of the hydrogel layer, the second side of the hydrogel layer, the body of the hydrogel layer comprises cells of the same or different types.

In a particular embodiment, the cells form one or more layers.

The cells include human cells. The human cell may be selected from primary cells, immortalized cell lines, epithelial cells, endothelial cells, mesenchymal stem cells, brain cells, muscle cells, immune cells, induced pluripotent stem cells, embryonic stem cells. All adherent cells presenting integrin receptors on the surface can be seeded and attached to both surfaces (i.e. the first and second side) of the hydrogel layer. Non-adherent cells and adherent cells may be encapsulated in the body of the hydrogel.

In particular embodiments, the device may be used to mimic mesenchyme, where mesenchymal-derived cells such as, but not limited to, fibroblasts, bone marrow-derived mesenchymal primary cells and/or intestinal stromal cells are susceptible to resident and migrating (e.g., flow-through or resident).

In one example, TC7 CaCo2 epithelial cells were seeded on the first side of the hydrogel layer, and HUVECs (human umbilical vein endothelial cells) were seeded on the second side of the hydrogel layer.

In a particular embodiment of the invention, the above microfluidic device is an integrated device comprising:

a hydrogel layer having first and second sides opposite each other,

wherein the hydrogel layer is interposed between a first element and a second element on a given axis substantially perpendicular to the hydrogel layer, and wherein the first element, the second element and the hydrogel layer have a determined shape and size, at least one microchannel is delineated at the first element and a first side of the hydrogel layer, and at least one cavity is between the second element and a second side of the hydrogel, the at least one microchannel and the at least one cavity being arranged relative to each other such that the given axis intercepts both the at least one microchannel and the at least one cavity; and

wherein the hydrogel layer is interposed between a first element and a second element on a given axis, the given axis being substantially perpendicular to the hydrogel layer, and the first element, the second element and the hydrogel layer have a shape and dimensions determined so as to define at least one microchannel between the first element and a first face of the hydrogel layer, and to define and/or form at least one cavity between the second element and a second face of the hydrogel, the at least one microchannel and the at least one cavity being arranged relative to each other such that the given axis intercepts the at least one microchannel and the at least one cavity, and,

wherein the integrated device comprises means for creating a pressure difference between the at least one microchannel and the at least one cavity.

The device may include a pump, valves and conduits to transfer liquid from the storage unit or gas from the compressed gas cylinder to the at least one microchannel and the at least one cavity. The device may further include a flow meter, a pressure gauge, a sensor, an indicator, and a switch.

The device may comprise a conduit (conduit) passing through the first element and/or the second element for introducing and/or withdrawing a liquid or gas into and/or from the at least one microchannel and/or the at least one cavity.

In a particular embodiment, the microfluidic device further comprises a conduit passing through the first element and the hydrogel layer to introduce and/or withdraw a liquid or gas into and/or from the at least one cavity.

Preferably, the connection to the conduit is made on the same side of the microfluidic device.

In particular embodiments, at least one of the first element and the second element is transparent to at least one electromagnetic wavelength to be emitted by the at least one microchannel and/or a component (e.g., a biological, chemical, or biochemical compound) within the at least one cavity to enable visual or signal detection, e.g., using a microscope.

In a particular embodiment, the first element, the second element and the hydrogel layer are mounted on a fixture, between a seat of the fixture and a clamping element, preferably removable, such as a nut.

The invention also relates to a method for producing a microfluidic device according to the invention, comprising:

a) producing or providing a first component and a second component;

b) functionalizing a surface of a first element (e.g., PDMS) with molecules comprising more than one chemical functional group;

c) optionally, functionalizing the surface of the second component (e.g., PDMS) with molecules comprising more than one chemical functional group;

d) producing or providing a hydrogel layer comprising one or more chemical functional groups effective to react with at least one chemical functional group of a molecule on the surface of the first element or the second element or both;

e) placing a hydrogel layer between the first element and the second element; and

f) allowing at least one chemical functional group of a molecule on the surface of the first member, the second member, or both members to covalently react with at least one chemical functional group in the hydrogel layer.

In another embodiment of the invention, the method for producing the microfluidic device of the invention comprises the steps of:

a) producing or providing a first part of a first element, a second part of a first element and a second element;

b) functionalizing a surface of a first component (e.g., PDMS) of a first member with molecules comprising one or more chemical functional groups;

c) optionally, functionalizing the surface of the second component (e.g., PDMS) with molecules comprising more than one chemical functional group;

d) producing or providing a hydrogel layer comprising one or more chemical functional groups effective to react with at least one chemical functional group of molecules on a surface of the first part and/or the second part of the first element;

e) placing a hydrogel layer between the first element and the second element; and

f) allowing at least one chemical functional group of a molecule on a surface of the first part of the first element and/or the second element to covalently react with at least one chemical functional group of the hydrogel layer.

In yet another embodiment of the invention, the method for producing the microfluidic device of the invention comprises the steps of:

a) producing or providing a first component, a first part of a second component and a second part of the second component;

b) functionalizing a surface of a first component (e.g., PDMS) with molecules comprising more than one chemical functional group;

c) optionally, functionalizing the surface of the first part of the second element (e.g. PDMS) with molecules comprising more than one chemical functional group;

d) producing or providing a hydrogel layer comprising one or more chemical functional groups effective to react with at least one chemical functional group of molecules on a surface of a first component of a first element and/or a second element;

e) placing a hydrogel layer between the first element and the second element; and

f) allowing the at least one chemical functional group of the molecule on the surface of the first component of the first element and/or the second element to covalently react with the at least one chemical functional group of the hydrogel layer.

In a particular embodiment, the method further comprises, between said steps d) and e), the steps of:

i) producing or providing at least one cell adhesion molecule, in particular a cell adhesion protein;

ii) optionally, during the step of producing the hydrogel layer, allowing at least one cell adhesion molecule to covalently bind to at least one chemical functional group of the hydrogel layer such that the at least one cell adhesion molecule is present in the bulk of the hydrogel layer;

iii) optionally, in the step of producing the hydrogel layer, seeding cells in the body of the hydrogel layer;

and, after step f), performing the steps of:

g) covalently bonding at least one cell adhesion molecule and the at least one chemical functional group of the hydrogel layer such that the at least one cell adhesion molecule is present on the first side, the second side, or both sides of the hydrogel layer; and

h) seeding the same or different types of cells on the first side, the second side, or both sides of the hydrogel layer.

The term "functionalized" refers to the attachment of a molecule, compound or atom to the surface of a material by the formation of a covalent bond.

As used herein, the term "functionalization" refers to a process of modifying the surface properties of a material, for example, by adding new functions via attachment of molecules or by substituting chemical bonds by functional groups.

In one embodiment of the invention, the surface of the first component, the second component, or both components is treated with oxygen plasma. In another embodiment, the surface of the first part and/or the second part of the first element or the surface of the first part and/or the second part of the second element is treated with oxygen plasma. An oxygen plasma is performed to introduce polar surface groups, such as silanol groups (SiOH). The modified treated surface is "activated" by oxygen plasma treatment to improve surface adhesion properties. One skilled in the art will be able to determine the parameters and conditions suitable for oxygen plasma treatment in order to modify a given material. For example, the surface of PDMS can be modified by exposure to oxygen plasma of 35mbars, 50mW for 1 minute.

In a specific embodiment, the oxygen plasma treated surface of the first member, the second member, or both members is further contacted with a molecule comprising a thiol or sulfhydryl functional group. This step involves grafting or attaching the molecule to the oxygen plasma treated surface. The molecule may be included in a solution at a concentration in the range of 0.1 to 10% (v/v).

The molecule may have the general formula X (CH)₂) n SiY3, containing reactive functional groups X and Y. X is a functional group (e.g., a thiol group) exposed on the surface of the first member, the second member, or both members that is capable of chemically reacting, particularly with a functional group of the hydrogel layer (e.g., a vinyl sulfone group) to form a covalent bond; n is an integer of 1 to 3; y is methoxyAnd ethoxy, methyl and the like. Y is a group bound to the surface of the first member or the second member. Examples of such molecules include, but are not limited to, (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) triethoxysilane, and (3-mercaptopropyl) methyldimethoxysilane. In a particular embodiment, the molecule comprises or consists of (3-mercaptopropyl) trimethoxysilane (MPS).

Techniques that may be used to contact molecules with the oxygen plasma treated surface of the first member, the second member, or both include, but are not limited to, dipping (i.e., a solution bath), dropping, spin coating, dip coating, vapor deposition.

In a specific embodiment, the oxygen plasma treated surface of the first member, the second member, or both members is immersed for one hour in a solution of 1% (v/v) (3-mercaptopropyl) trimethoxysilane (MPS) in a mixture of ethanol and acetic acid. The MPS treated surface was then washed with 70% (v/v) ethanol and baked at 110 ℃ for 1 hour.

In a specific embodiment, the functionalized surfaces of the first element, the second element, or both elements are incubated in a 10mM solution of Dithiothreitol (DTT). This step ensures reduced disulfide bonds on the functionalized surface. The surface was then rinsed with pure water and dried with compressed air.

In a preferred embodiment, the hydrogel layer is produced by crosslinking a vinylsulfone functionalized polyethylene glycol (PEG-VS) macromer and a thiol functionalized polyethylene glycol (PEG-SH) macromer. The crosslinking is initiated by mixing stock solutions of the PEG macromers (PEG-VS and PEG-SH) using a predetermined stoichiometric ratio suitable for preparation, e.g., vinyl sulfone can be added in a molar excess of 1.2mM compared to thiol. The resulting gel-like mixture is then transferred to a suitable carrier material and contacted with a flat hydrophobic material (e.g., a hydrophobic slide). Once the desired degree of polymerization or crosslinking has been achieved, for example, by a michael-type addition reaction between vinyl sulfone groups and thiol groups, the resulting hydrogel is then cured at room temperature. The duration of polymerization or crosslinking depends on the concentration of PEG. Prior to completion of gelation, the hydrogel is transferred to the first or second element (e.g., a surface-activated PDMS body) to cover the one or more grooves included therein, thereby forming one or more microchannels.

In another embodiment, PEG macromers (including PEG-VS macromers and/or PEG-SH macromers) are crosslinked by a polypeptide to create a hydrogel layer. The hydrogel layer comprising polypeptides as cross-linkers is sensitive to various proteases, including metalloproteinases (MMPs).

In a specific embodiment, at least one cell adhesion protein is attached to the first side, the second side, or both sides of the hydrogel layer (particularly by a heterobifunctional protein crosslinking reagent). The attachment of the cell adhesion protein may be performed as follows: a liquid containing the cell adhesion protein is perfused into the microchannel and/or cavity, followed by an incubation period. The cell adhesion protein is preferably coupled to a heterobifunctional protein crosslinking agent that reacts with surface functional groups on the first, second, or both sides of the hydrogel layer.

The stoichiometric ratio of the cell adhesion protein to the heterobifunctional protein cross-linking agent is in the range of 1:1 to 1: 20, or more.

In a specific embodiment, at least one cell adhesion protein is attached (particularly via a heterobifunctional protein cross-linker) to the thiol group of the PEG-SH macromer prior to cross-linking with the PEG-VS macromer.

Cell adhesion proteins include recombinant domain fragments of fibronectin, such as FN9-10 fragments.

The heterobifunctional protein crosslinking agent includes an amine-to-sulfhydryl crosslinking agent, such as NHS-PEG-maleimide crosslinking agent.

In one example of the invention, the maleimide groups of the NHS-PEG-maleimide crosslinker are reacted with thiol groups (-SH) on the first, second, or both sides of the hydrogel layer.

In a specific embodiment, the cells are seeded by sequentially delivering or injecting the cells suspended in a liquid through a microchannel or cavity to the first or second side of the hydrogel layer, followed by an incubation period.

In a specific embodiment, the cells are seeded in the body of the hydrogel by adding the cells to a mixture of PEG-VS macromer and PEG-SH macromer attached to at least one cell adhesion molecule.

The cell adhesion molecules, in particular cell adhesion proteins, are advantageously modified by Fc antibody fragments or by heterofunctional protein crosslinkers. The Fc antibody fragment is adsorbed (e.g., non-covalently linked) to a hydrogel layer containing protein a or protein G or any other molecule having affinity for the Fc fragment. Protein a or protein G or any other molecule with affinity for the Fc fragment may be incorporated into the hydrogel layer during the hydrogel synthesis step (e.g., may be added to a mixture of PEG macromonomers).

The heterofunctional protein crosslinking reagent is attached to at least one functional group (particularly a thiol group) of the hydrogel layer by forming a covalent bond between the heterobifunctional protein crosslinking reagent and the at least one functional group (particularly a thiol group) of the hydrogel layer.

In a specific embodiment, prior to completion of crosslinking of the hydrogel layer, contacting the hydrogel layer comprising one or more chemical functional groups with a surface of a first element, a second element, or both elements comprising molecules having one or more chemical functional groups to initiate a chemical reaction, wherein at least one chemical functional group of the hydrogel layer forms a covalent bond with at least one chemical functional group of the molecules at the surface of the first element, the second element, or both elements.

The invention also relates to a method for driving a hydrogel layer of a microfluidic device of the invention, the method comprising:

a) introducing a liquid or gas into at least one microchannel between the first element and the first side of the hydrogel layer;

b) introducing a liquid or gas into at least one cavity between the second element and the second side of the hydrogel layer;

c) flowing the liquid or gas through the at least one microchannel between the first element and the first side of the hydrogel layer;

d) flowing the liquid or gas through the at least one cavity between the second element and the second side of the hydrogel layer; and

e) adjusting or changing the flow rate of the liquid or the pressure of the gas in the at least one microchannel or the at least one cavity creates a pressure differential between the at least one microchannel and the at least one cavity, expanding or contracting the hydrogel layer by flexing or bending alternately in two opposite directions perpendicular to the plane of the hydrogel layer toward the at least one microchannel or the at least one cavity.

According to the present invention, the microfluidic device may be used as an organ chip device, in particular a device that includes human cells and simulates a physiological condition. Accordingly, as shown in the embodiments, the driving of the hydrogel layer may be achieved, in particular using the following steps and/or parameters.

In a particular embodiment, the fluid pressure differential is applied by applying different flow rates of liquid in the microchannel (i.e., between the first element and the first side of the hydrogel layer) and liquid in the cavity (i.e., between the second element and the second side of the hydrogel layer). In one embodiment, only the flow rate of the liquid in the microchannel is changed, the flow rate of the liquid in the cavity being kept constant or static at atmospheric pressure.

In a specific embodiment, the flow rate of the liquid in the microchannel is 0 μ l.h^-1To 10,000 μ l.h^-1Within the range of (1). In a preferred embodiment, the flow rate of the liquid in the microchannel is 30 μ l.h^-1To 1500 mu l.h^-1. In one embodiment, the flow rate is periodically at 30 μ l.h^-1To 1500 mu l.h^-1With a frequency of up to 0.2 Hz. In another embodiment, the microchannel is at 1500 μ l.h^-1A pulsed flow of the liquid is applied for 5 seconds per minute.

In a particular embodiment, the stretching of the hydrogel layer is brought about up to 25%, in particular 10%, which is brought about in particular by: the flow rate in the microchannel was changed from 30. mu. l.h in 10 seconds^-1Periodically increased to 1500 mu l.h^-1Then a relaxation time of 10 to 50 seconds is carried out at 30 μ l.h-1. This actuation of the hydrogel layer may take several days, for example 7 days.

In a specific embodiment, the liquid in the microchannel and the liquid in the cavity are the same or different.

Specific features of the invention will be illustrated in the following figures and examples. The features disclosed herein also define the embodiments of the invention as described above.

Drawings

FIG. 1: (a-B) the concept of a microfluidic device based on a Hydraulically driven hydrogel layer (HAHL); the thin layer of hydrogel is driven over time by the pressure differential between the upper and lower channels. Actuation is possible due to the covalent binding of the hydrogel to the first element. The drive profile is directly controlled by the user of the HAHL device by the hydraulic pressure which varies with time. (C) As an early proof of concept, the HAHL device was seeded with Caco2/E-Cad:: GFP epithelial cells in the upper channel (P1) and wild type HUVEC in the lower channel (P2). The scale is 200 microns. Dotted line rectangle: the insertion position. Dotted line: the upper microfluidic channel limit. (D) Preliminary results for early HAHL microfluidic devices. The closed circuit was used for the assessment of HAHLs perfusion and hydrogel layer deformation over several days, and cells placed on top of the driven hydrogel were observed using a 2D microscope. (E) Deformation of PEG-fibronectin (PEG-Fn) mechanical sensors was evaluated based on microscopic analysis of periodic cell displacement. Error bars-standard deviation over 3 features. Visualization of C2C12 cells growing two-dimensionally on the PEG-Fn hydrogel layer. (F) Differential deformation in two regions of the grafted hyaluronic acid-gelatin hydrogel on a thin PEG mechanical sensor. The star indicates a point where the circular distortion is so great as to cause the beads above the center of the channel to move away from the focal plane, thus forcing the use of a point that no longer has a small angular approximation. R2 is the coefficient of determination of the linear regression. Error bars-standard deviation based on analysis of 5 features (paired beads).

FIG. 2: the relationship between flow rate and mean cell area deformation was quantitatively evaluated. Panels a and B show two photographs of cells mechanically stimulated on a HAHL device at different flow rates. The trend line is calculated as a logarithm due to the way stretching occurs in the material.

FIG. 3: epithelial differentiation on HAHL-microfluidic device. As early as 2 days after seeding, the seeded CaCO2 cells showed evidence of clear 3D tissue (organization) formation of villous structures (stretched condition: under continuous flow). The same cells will still experience 24 hours delayed 3D tissue.

FIG. 4A: a three-dimensional cross-sectional view of the microfluidic device according to the present invention through the plane of the microchannel and cavity.

FIG. 4B: fig. 4A shows an exploded view of the components of the device.

FIG. 5: cross-sectional view of an exemplary embodiment of a first element of a microfluidic device of the present invention.

FIG. 6: various device configurations (cross-sectional views) of the present invention.

Detailed Description