Biochip for cell culture

Abstract

A biochip for cell culture comprising a growing chamber comprising a hydrogel network and at least one configuration element.

Description

BIOCHIP FOR CELL CULTUREFIELD OF INVENTION

[0001] The present invention relates to a biochip for cell culture.BACKGROUND OF INVENTION

[0002] Cell cultures are used today as a tool for the toxicological evaluation of substances. Bioreactors reproduce an environment favorable to the development and organization of cells, close to that of a tissue or an animal or human organ. Thanks to them, one can predict the reaction of an organ to a substance such as a xenobiotic, a cosmetic, a drug or more generally any active principle, and develop relevant in vitro models. These models are increasingly used in pharmaceutical research as they represent a serious alternative to in vivo models, z.e., animal experimentation, against which both economic and ethical pressures are emerging at the international level. Cell culture also represents the basis for the creation of artificial organs capable of replacing failing or absent organs, one of the challenges of tissue engineering.

[0003] Some in vitro technics using Petri dishes are known. However, these culturing methods leads to low predictive accuracy.

[0004] Other known methods use biochips leading to a dynamic cell culture which can be miniaturized and parallelized. The biochips include at both ends of a culture chamber a fluid inlet and a fluid outlet to allow the passage of a nutritive fluid necessary for the development of cells. Various biochips have been proposed for this purpose. However, despite the higher predictive accuracy compared to Petri dishes, these known systems are not satisfactory. Indeed, the use of biochips leads to low throughput.

[0005] To increase the physiology, three-dimensional (3D) structures are created in the biochip chamber so that cells are able to grow in 3D. To do so, the known biochips comprise an upper surface and a lower surface wherein the structures are manufactured, for example by molding or material removal. However, the manufacturing of such abiochip comprising structures is challenging because the slightest shift between the upper surface and the lower surface of the biochip leads to a decay of the fluid circulation efficiency. To avoid this problem, the biochips are molded in one piece in silicone. However, silicone has chemical, physical and mechanical properties leading to the impossibility to configure the properties of the biochip according to the cell to be cultured. Moreover, some of the known biochips comprise molded structures However, the cultured cells are growing only along the structure, therefore, the cells do not grow to fill, the whole volume of the growing chamber. Even complex micro manufacturing does not allow the formation of a structure that allows the cells to homogeneously fill the growing chamber.

[0006] There is thus a need for a biochip which is cheap and easy to manufacture and which increases the efficiency of the fluid circulation and the 3D growth of the cultured cells.

[0007] To this end, a purpose of this invention is to provide a biochip comprising a hydrogel allowing the creation of 3D culturing support after the assembling of the upper and lower surface of the biochip. Moreover, the biochip comprises at least one configuration element allowing to personalize the growing chamber, optimize the cell growth and / or ease the manufacturing of the biochip.SUMMARY

[0008] This invention thus relates to a biochip for cell culture comprising an upper surface, a lower surface, at least one inlet and at least one outlet, each inlet and each outlet being disposed in the upper surface and / or the lower surface, wherein the upper surface and the lower surface are configured in order to form a growing chamber between the upper surface and the lower surface when the upper surface is abutted on the lower surface; wherein the growing chamber allows fluidic communication from the at least one inlet to the at least one outlet; wherein the growing chamber comprises a hydrogel network; andwherein the growing chamber comprises at least one of the following configuration elements: a set of three-dimensional structures disposed on the upper surface and / or the lower surface; an intermediate layer disposed between the upper surface and the lower surface; or a spacer disposed between the upper surface and the lower surface, the spacer forming walls of the growing chamber, the upper surface and the lower surface closing the growing chamber.

[0009] Indeed, the use of a biochip allows parallelization and fluid recirculation. Moreover, the biochip is easy to use and allows a multi-organs culture.

[0010] Thanks to the inlet and the outlet, the biochip is compatible with the existing laboratory materials. Moreover, the biochip allows a continuous observation, for example microscopic observation, through its walls without need to stop the culture process.

[0011] The use of a hydrogel allows to locally modify the chemical, physical, and / or mechanical properties of the biochip. This allows to increase the growth of the cells thanks to the adaptation of the biochip properties to the cell to be cultured. Moreover, this allows to create a culturing environment which is close to the in vivo environment since the hydrogel can mimic the physical, mechanical and chemical properties of an organ onto - or into - which the cells should grow. Furthermore, the hydrogel network may allow to efficiently culture several types of cells by creating a network composed of a plurality of surfaces presenting different properties at different locations of the growing chamber. In other words, the hydrogel behaves as a three-dimensional scaffold, potentially with locally modified physicochemical properties.

[0012] Moreover, the polymerization of the hydrogel may be performed after the assembling of the upper and the lower surface thus facilitating the manufacture of the biochip.

[0013] Finally, the insertion of a configuration element allows to personalize the growing chamber, optimize the cell growth or ease the manufacturing of the biochip.

[0014] According to an advantageous aspect of the invention, the growing chamber comprises the set of three-dimensional structures

[0015] Indeed, the geometry of the set of three-dimensional structures is predetermined which allows to adjust the way the cells are growing by guiding the creation of the hydrogel network along a predetermined geometry. In other words, the set of three- dimensional structures locally modify the geometry of the hydrogel network providing additional mechanical and physical constraints.

[0016] Moreover, the set of three-dimensional structures provides a support for the hydrogel network so that the network is less subject to damages by the fluid circulation.

[0017] Advantageously, the three-dimensional structures are only present on the lower surface, the upper surface being flat. This improves the circulation of the fluid from the inlet to the outlet and prevents the need of a perfect alignment between the structures of the upper and the lower surfaces.

[0018] The use of a hydrogel network built around the three-dimensional structures allows to protect the cells from damaging by contact with the three-dimensional structures which may have sharp edges.

[0019] According to an advantageous aspect of the invention, the set of three- dimensional structures forms a structural network between the at least one inlet and the at least one outlet.

[0020] According to an advantageous aspect of the invention, a total volume of three- dimensional structures computed as a sum of a volume of each three-dimensional structure of the set of three-dimensional structures is ranging from 5% to 50% of the volume of the growing chamber.

[0021] This volume is small enough to provide a support for the hydrogel network wherein a good fluid circulation is achievable without need of increasing the fluid pressure which may damage the cells. Moreover, this volume occupied by the three- dimensional structures is large enough to obtain a sufficient volume of hydrogel network for growing cells.

[0022] According to an advantageous aspect of the invention, each three-dimensional structure is a pit or a channel in the upper surface and / or the lower surface.

[0023] According to an advantageous aspect of the invention, each three-dimensional structure is crescent- shaped, each three-dimensional structure comprising a concave surface oriented towards the at least one inlet of the bioship and a convex surface oriented towards the at least one outlet of the bioship, the set of three-dimensional structures being arranged along a determined pattern.

[0024] The hydrogel network preferably fills the volume defined by the concave surfaces therefore better controlling the density of the network. This allows the cells to preferably nestle on the side of the concave surface in order to grow while receiving the flow of nutrient. Indeed, contrarily to the convex surface, the cells on the concave surface are less damaged by the fluid currents.

[0025] According to an advantageous aspect of the invention, each crescent- shaped three-dimensional structure has a length ranging from 100 pm to 900 pm and a width ranging from 150 pm to 450 pm, preferably each crescent-shaped three-dimensional structure ends with two rounded tips.

[0026] The rounded tips avoid damaging the cells in case of absence of hydrogel network along at least one of the tips of the crescent-shaped three-dimensional structures.

[0027] According to an advantageous aspect of the invention, the determined pattern forms at least two rows, each row comprising at least two crescent-shaped three- dimensional structures.

[0028] The determined pattern allows to obtain a controlled structure which is well defined when manufacturing the biochip. This thus allows to define specific features for the flow configuration such as stopping points by a surface, bypassed zone, etc.

[0029] According to an advantageous aspect of the invention, a distance between the centers of two successive crescent-shaped structures of the same row is ranging from 20 pm to 500 pm.

[0030] This allows a stable fluid circulation without need to increase the fluid pressure.

[0031] According to an advantageous aspect of the invention, each three-dimensional structure is in contact with the upper surface and the lower surface.

[0032] This allows to take advantage of the whole height of the growing chamber. The growing of cells is thus more efficient.

[0033] According to an advantageous aspect of the invention, the growing chamber comprises the intermediate layer.

[0034] The intermediate layer allows to fix evenly the upper surface and the lower surface.

[0035] Moreover, the intermediate layer may be configured to homogenize and stabilize the fluid circulation thanks to a controlled porosity of the intermediate layer. The parameters of the fluid circulation through the cells are thus better controlled. The intermediate layer thus allows to protect the structures and the cells inside the biochip.

[0036] Finally, the intermediate layer may also be configured to receive a cell culture allowing to co-culture different types of cells. The intermediate layer thus allows to physically separate the two surfaces while allowing the exchange of fluid and bioactive molecules.

[0037] According to an advantageous aspect of the invention, the upper surface, the lower surface and the intermediate layer are bound together on a binding area representing less than 5% of an area of the upper surface, preferably the binding area is on the periphery of the biochip.

[0038] This low binding area advantageously allows the reduction of costs when manufacturing the biochip. Moreover, it allows an easier destruction of the biochip in order to recover the three-dimensional structures and / or hydrogel for biological, mechanical and / or chemical analysis.

[0039] According to an advantageous aspect of the invention, the biochip further comprises the spacer.

[0040] The presence of a spacer is advantageous because it allows to space out the upper surface and the lower surface thereby creating a volume between the upper surface and the lower surface forming the growing chamber. In other words, the spacer forms the walls (or border) of the growing chamber, the upper surface and the lower surface closing the growing chamber from the top and the bottom. There is thus no need of grooving the upper and lower surfaces therefore facilitating the manufacture of the biochip.

[0041] The spacer is further advantageous because it allows to combine several physical characteristics according to the different material used for the upper and lower surfaces and for the spacer. For example, the spacer may have sealing properties allowing to avoid any leakage when the upper surface and the lower surface are abutted, the spacer being disposed between the upper and lower surfaces.

[0042] Finally, the spacer increases the surface of contact for the hydrogel matrix. It can also contribute to create locally different micro environments for the formation of the hydrogel matrix, for example a heterogeneous effect in the density of the hydrogel.

[0043] According to an advantageous aspect of the invention, the growing chamber comprises a coating on at least one of the surfaces of the growing chamber.

[0044] The coating may be a thin layer deposited inside the growing chamber. Such a coating does not increase the total height of the biochip but provide particular chemical and / or physical properties to the surfaces onto which it is deposited.

[0045] For example, the coating is deposited on the upper and / or lower support, before formation of the hydrogel network.

[0046] Preferably, the coating is deposited on the surfaces of the growing chamber after the hydrogel network has been created. Such a coating deposition allows to modify the chemical and / or physical properties of the whole growing chamber, z.e., the hydrogel network and the surfaces of the upper and lower support that are not already covered by the hydrogel network.

[0047] The invention also relates to a manufacturing process of the biochip, the process comprising:Injecting a polymer formulation in the growing chamber;Forming the hydrogel network in three dimensions from the polymer formulation.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 shows an image of a biochip according to an embodiment of the invention.

[0049] Figure 2 shows a side-view of a biochip according to an embodiment of the invention.

[0050] Figure 3 shows a side-view of a biochip comprising an intermediate layer.

[0051] Figure 4 shows a side-view of a biochip comprising a spacer and a coating.

[0052] Figure 5 shows a top-view of a determined pattern for a set of three-dimensional structures.

[0053] Figure 6 is a combination of graphs. Figure 6A shows a detailed image of the set of three rows of crescent- shaped three-dimensional structures substantially perpendicular to the direction of the length of the upper and lower surfaces. Figure 6B shows a detailed image of the set of three rows of crescent- shaped three-dimensional structures substantially parallel to the direction of the length of the upper and lower surfaces. Figure 6C shows a detailed image of a crescent-shaped three-dimensional structure.

[0054] Figure 7 is a combination of graphs. Figure 7A shows a view of the upper surface and lower surface of a known biochip of the prior art. Figure 7B shows a view of the upper surface and lower surface of the biochip according to an embodiment of the invention.

[0055] Figure 8 is a microscopy picture of the cells that have grown on the hydrogel network inside a growing chamber comprising a set of three-dimensional structures.

[0056] Figure 9 is a microscopy picture of the cells that have grown on a set of three- dimensional structures without hydrogel network.DETAILED DESCRIPTION

[0057] The following detailed description will be better understood when read in conjunction with the drawings. For the purpose of illustrating, the device is shown in the preferred embodiments. It should be understood, however that the application is not limited to the precise arrangements, structures, features, embodiments, and aspect shown. The drawings are not drawn to scale and are not intended to limit the scope of the claims to the embodiments depicted. Accordingly, it should be understood that where features mentioned in the appended claims are followed by reference signs, such signs are included solely for the purpose of enhancing the intelligibility of the claims and are in no way limiting on the scope of the claims.

[0058] This invention relates to a biochip 100 for cell culture.

[0059] A cell culture is the process by which cells are grown ex vivo under controlled conditions. This allows to reproduce a cell structure close to that of a tissue of an animal or human organ in order to perform experiments, analysis and evaluation outside the human or animal body. To do so, cell cultures are seeded in a growing (culturing) chamber 30 and a flow of fluid is set through the growing chamber 30. The fluid is preferably a nutritive fluid but may also comprise bioactive molecules in order to study the reaction of the cells to these molecules.

[0060] The biochip 100 is illustratively represented in figures 1-4.

[0061] As shown in figures 2-4, the biochip 100 comprises an upper surface 10 and a lower surface 20. The surfaces (10, 20) may be made in any suitable material. A suitable material may be moldable, injectable and / or printable by 3D printing. The upper surface 10 and the lower surface 20 may be made of different material. This isadvantageous to locally modify the chemical, physical and / or mechanical properties of the biochip. Preferably, the surfaces (10, 20) are made in material not absorbing the molecules. The surfaces (10, 20) may be made in gas permeable material. For example, the surfaces (10, 20) are permeable to oxygen and / or carbon dioxide. The surfaces (10, 20) may be rigid (i. e. , leading to fracture when subjected to torsion or bending) or flexible. Preferably, the surfaces (10, 20) are transparent in order to allow visual inspection of the cell culture.

[0062] Suitable material for the surfaces (10, 20) are selected in the group of Silicone, in particular Polydimethylsiloxane (PDMS), Perfluoropolyether (PFPE), Poly(methyl methacrylate) (PMMA) or Polycarbonates (PC), polymethylpentene (PMD). PFPE is advantageous because it is colorless, odorless and completely inert in the presence of most chemical agents, especially oxygen. PMMA or PC are advantageous because of their low absorption of molecules, transparency and because they are suitable for injection technologies of manufacturing. PDMS is advantageous because it is transparent, inert and non-toxic. In addition, PDMS is very easy to mold in laboratory operations.

[0063] The upper and lower surfaces (10, 20) may be made by molding or additive manufacturing such as 3D printing.

[0064] As shown in figure 2, each surface (10, 20) extends along a longitudinal plan P defining a length and a width for each surface. As shown in figures 2-4, the surfaces (10, 20) also extend along an axis A perpendicular to the longitudinal plan P so that each surface (10, 20) has a height measured along the perpendicular axis A. Each surface (10, 20) has two longitudinal faces (11, 13, 21, 23) linked by a border (Bl, B2). Preferably, the longitudinal faces (11, 13, 21, 23) are parallel to the longitudinal plan P while the borders (Bl, B2) are parallel to the perpendicular axis A.

[0065] Preferably, the upper and lower surfaces (10, 20) are shaped so that they can be superposed in a way that their longitudinal plans P are parallel. More precisely, they are configured so that one of the longitudinal faces (13, 23) of each surface are abutted against each other. The upper and lower surfaces (10, 20) preferably present a mirror shape so that, when abutted against each other, an interfacing plan I formed between thetwo abutted faces (13, 23) - hereafter named the interior faces (13, 23) - is a symmetry plan between the two surfaces (10, 20).

[0066] The upper surface 10 and the lower surface 20 are configured in order to form a growing chamber 30 when the upper surface 10 is abutted on the lower surface 20. For example, the interior faces (13, 23) comprise at least one groove. When abutted, the groove in one interior face (13, 23) may form, with the opposite interior face (23, 13) or with the groove in the opposite interior face (23, 13), a single chamber, a plurality of chambers, micro-channels or micro-holes in fluidic communication. Preferably, the grooves in the interior faces (13, 23) are disposed in order to be superposed when abutted, preferably in a mirror manner relatively to the interfacing plan I.

[0067] The height of the growing chamber 30, measured as the largest distance between the interior faces (13, 23) along the perpendicular axis A when the upper surface 10 and lower surface 20 are abutted, may range from 50 pm to 500 pm, preferably from 100 pm to 300 pm. The height of the growing chamber 30 corresponds, when grooves are present, to the sum of the largest height of the grooves in each face (13, 23). The height of the groove in one of the surfaces (10, 20) may be larger than the groove in the other surface (20, 10).

[0068] The biochip 100 also comprises at least one inlet 1 and at least one outlet 5. For a set comprising one inlet 1 and one outlet 5, the inlet 1 and the outlet 5 may be disposed both in the upper surface 10 as in figures 1-4, but also both in the lower surface 20 or one in the upper surface 10 and the other in the lower surface 20. For two sets, each set comprising one inlet 1 and one outlet 5, one of the inlets 1 and one of the outlets 5 may disposed both in the upper surface 10 while the other inlet 1 and the other outlet 5 may disposed both in the lower surface 20. Disposing the inlet 1 and outlet 5 of the same set in different surfaces (10, 20) is advantageous for specific flow requirement. The inlet 1 and outlet 5 allows a fluidic communication between the exterior of the biochip 100 and the growing chamber 30. Each of the inlet 1 and the outlet 5 thus forms a channel or pit through the upper or the lower surface (10, 20). More precisely, the inlet 1 and outlet 5 unclog on the face (11, 21) - hereafter named the exterior surface (11, 21) - opposite to the interior face (13, 23) or on the border (Bl, B2).

[0069] The inlet 1 and outlet 5 may comprise a wall extending away from the exterior surface (11, 21). The inlet 1 and outlet 5 thus form a plug for easily connecting a fluidic system. In one embodiment, the inlet 1 and outlet 5 are intrinsically comprised in the upper and lower surfaces (10, 20) in order to be irreversibly bound to the upper and lower surfaces (10, 20). The inlet 1 and outlet 5 may be made in a single piece with the upper and lower surfaces (10, 20).

[0070] In an alternative embodiment, the inlet 1 and outlet 5 are reversibly bound to the upper and lower surfaces (10, 20), for example by screwing. The reversible binding is advantageous because if the inlet 1 or the outlet 5 is defective, it may be replaced without need to replace the whole surface (10, 20). Advantageously, the inlet 1 and outlet 5 are made in a flexible material, for example in polypropylene to be less subject to damages. Preferably, the inlet 1 and outlet 5 are standard ports of 4 mm of diameter and 10-32 UNF.

[0071] The growing chamber 30 allows fluidic communication from the inlet(s) 1 to the outlet(s) 5. For example, the growing chamber 30 is in communication with the inlet(s) 1 and the outlet(s) 5 thought a connecting network 35 as shown in figures 5 or 8B. The connecting network 35 may be grooved in the surfaces (10, 20) so as the growing chamber 30. Preferably, the inlet(s) 1 and the outlet(s) 5 are positioned to set a flow inside the growing chamber 30 in the direction of the length of the upper and lower surfaces (10, 20).

[0072] The growing chamber 30 comprises a hydrogel network 31. Using a hydrogel network 31 is advantageous because it allows to locally modify the chemical, physical, and / or mechanical properties of the biochip. This allows to increase the growth of the cells thanks to the adaptation of the biochip properties to the cells to be cultured. Therefore, the cells preferentially nestle on the hydrogel network 31. Moreover, this allows to create a culturing environment which is close to the in vivo environment since the hydrogel network 31 can mimic the physical, mechanical and / or chemical properties of an organ onto - or into - which the cells should grow. Furthermore, the hydrogel network 31 may allow to efficiently culture several types of cells by creating a network composed of a plurality of surfaces presenting different properties at different locations inside the growing chamber. Moreover, the hydrogel network 31 allows the growth of thecultured cells in three-dimensions up to cover the overall height of the growing chamber therefore optimizing the volume of the cultured cells relatively to the longitudinal size of the growing chamber 30 compared to biochips comprising only three-dimensional structures. The hydrogel network 31 may comprise collagen, Matrigel™, alginate, polysaccharide, at least one mixture of extracellular matrix proteins, ... The hydrogel network 31 may take the form of a hydro-scaffold which associates the behavior of a solid scaffold and of a hydrogel.

[0073] The hydrogel network 31 may be manufactured by the manufacturing process of the invention. Firstly, after abutting the upper surface 10 on the lower surface 20 in order to form the growing chamber 30, a polymer formulation is injected in the growing chamber 30. Then, the hydrogel network 31 is formed in three dimensions from the injected polymer formulation.

[0074] The hydrogel network 31 has a volume ranging from 1% to 70%, preferably from 5% to 50%, of the volume of the growing chamber 30. By hydrogel volume, it is meant here the volume of the scaffold made of hydrogel, after removal of the formulation remaining after hydrogel formation: it corresponds more or less - depending on the amount of soluble compounds remaining in the formulation - to the dry matter percentage of the formulation leading to the hydrogel.

[0075] For example, if the hydrogel network 31 to be formed is a cryogel, the polymer formulation is injected in the form of an aqueous solution. The polymer formulation thus forms a continuous phase in the growing chamber 30. Then, the biochip 100 is cooled so that the hydrogel precursors precipitates / jellifies in order to form a network in three dimensions. This step thus allows a macromolecular organization of the hydrogel. The hydrogel is then polymerized or reticulated along the network. During this step, the hydrogel network 31 is created by differentiation from the continuous phase. At the end of the polymerization, there is thus, in the growing chamber 30, the hydrogel network 31 and the rest of continuous phase which has not polymerized. Finally, the continuous phase is removed from the biochip 100 for example by suction or by heating. Preferably, the mass fraction of hydrogel precursors in the formulation is ranging from 1% to 70%, preferably from 5% to 50%.

[0076] In another example, the polymer formulation is injected in the form of an aqueous solution. Then, the biochip 100 is heated in order to create the macromolecular organization of the hydrogel in the form of a network, either by polymerization / reticulation - for heat activated polymerizable compounds - or by precipitation / aggregation or by phase transition of the macromolecular - for temperature- controlled polymers. During this step, the hydrogel network 31 is created by differentiation from the continuous phase. Finally, the continuous phase is removed from the biochip 100 for example by suction. Preferably, the mass fraction of hydrogel precursors in the formulation is ranging from 1% to 70%, preferably from 5% to 50%.

[0077] In another example, the polymer formulation is injected in the form of an aqueous solution. The biochip is exposed to activation radiation, for instance to UV light. The hydrogel precursors undergo radical polymerization - usually initiated by initiators - and form a network. Finally, the remaining formulation is removed from the biochip 100 for example by suction. Preferably, the mass fraction of hydrogel precursors in the formulation is ranging from 1% to 70%, preferably from 5% to 50%. The use of a material transparent to the UV for the surfaces (10, 20) is required in this example.

[0078] In another example, a memory foam polymer formulation is injected in the form of an aqueous solution. Then, the biochip 100 is subjected to a mechanical constraint to form the hydrogel network 31. The mechanical constraint may be an external pressure applied on the biochip. The pressure may be homogenous along the surface of the biochip of may present local variations allowing to organize the hydrogel network along a predetermined distribution.

[0079] The growing chamber 30 further comprises at least one of the following configuration elements: a set of three-dimensional structures 32 disposed on the upper surface 10 and / or the lower surface 20; an intermediate layer 40 disposed between the upper surface 10 and the lower surface 20; or a spacer 50 disposed between the upper surface 10 and the lower surface 20.

[0080] The three-dimensional structures 32 provide a support for the hydrogel network 31 which is formed on each surface of the growing chamber, i.e., on the upper surface 10 and the lower surface 20 and also on the three-dimensional structures 32. Therefore, when a fluid is circulating through the hydrogel network 31, the network 31 is less subjected to damages by the stream of fluid. Even if the circulation of the fluid is mainly directed by the hydrogel network 31, the three-dimensional structures 32, since they locally modify the geometry of the hydrogel network 31, also allow to influence the fluid circulation. Moreover, locally modifying the geometry of the hydrogel network 31 allows to provide additional mechanical constraints to the biochip 100. Compared to a cells culture in a biochip comprising only three-dimensional structures, the use of a hydrogel network 31 allows a three-dimensional growth of the cells in the whole volume of the growing chamber instead of close to the surface of the three-dimensional structures. Moreover, the hydrogel network 31 provides a smooth support for the cells deposition and avoids the damage of the cells against the sharp edges of the three-dimensional structures 32.

[0081] Each three-dimensional structure 32 may comprise at least one pit, at least one channel, at least one microchamber, or a mix of at least one microchamber and at least one channel in the upper surface 10 and / or the lower surface 20. Each pit have an extremity opening towards the growing chamber 30. The pit has a height lower than the height of the upper surface 10 and / or the lower surface 20 into which it is formed. For example, the pit has a height ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The pit may have a diameter ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The pit may have a circular section, a polygonal section or an irregular section. The diameter of the pits may vary along its height. Each channel is adjacent to the surfaces of the growing chamber 30. The channel has a depth lower than the height of the upper surface 10 and / or the lower surface 20 into which it is formed. For example, the channel has a depth ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The channel may have a section ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The channel may have a circular section, a polygonal section or an irregular section. The section of the channel may vary along its length. Each microchamber is adjacent to the surfaces of the growing chamber 30. The microchamber has a depth lowerthan the height of the upper surface 10 and / or the lower surface 20 into which it is formed. For example, the microchamber has a depth ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The microchamber may have a section ranging from 10 pm to 1000 pm, preferably from 50 pm to 500 pm. The section of the microchamber may vary along its length. The microchamber may have a cylindrical volume or an irregular volume.

[0082] Alternatively, the set of three-dimensional structures 32 may form a structural network between the inlet 1 and the outlet 5 as shown in figures 1 and 7B. An example of a set of three-dimensional structures 32 is illustrated in figures 6A, 6B and 6C. The height of each three-dimensional structure 32, measured along the perpendicular axis A, is lower or equal to the height of the growing chamber 30. In one embodiment, the height of each three-dimensional structure 32 may range from 10 pm to 300 pm, preferably from 50 pm to 150 pm. In another embodiment, each three-dimensional structure 32 is in contact with the upper surface 10 and the lower surface 20. In other words, the height of each three-dimensional structure 32 is equal to the height of the growing chamber 30 at the position of said three-dimensional structure 32.

[0083] The three-dimensional structures 32 may be only present on one of the surfaces (10, 20), the other surface (20, 10) being flat, z.e., without channel or pit on its interior face 13. Similarly, the connecting network 35 may be only present on one of the surfaces (10, 20), the other surface (20, 10) being flat. For example, the three-dimensional structures 32 and / or the connecting network 35 may be only present on the lower surface 20, the upper surface 10 being flat. Figure 7 compares the upper and lower surfaces (10, 20) of a biochip according to the prior art (fig. 7A) and according to the invention (fig. 7B). Contrarily to the prior art, the interior face 13 of the upper surface 10 does not comprise any groove from the inlet 1 to the outlet 5 whereas the interior face 13 of the upper surface 10 of the biochip of the prior art comprises grooves between the growing chamber and the inlet / outlet. In the biochip of the priori art (fig. 7A), both the upper and lower surfaces (10, 20) comprise grooves. The grooves form the connecting network 35 when the surfaces (10, 20) are abutted. A flat upper surface 10 advantageously improves the circulation of the fluid from the inlet 1 to the outlet 5 andprevents the need of a perfect alignment between the grooves of the upper and the lower surfaces (10, 20).

[0084] Each three-dimensional structure 32 has a volume so that the sum of the volume of each three-dimensional structure 30, z.e., the total volume of the set of three- dimensional structures 32, is ranging from 5% to 50% of the volume of the growing chamber 30. When the three-dimensional structures 32 are in the form of a structural network, the hydrogel network 31 is preferably comprised between the three-dimensional structures 32. In this embodiment, the sum of the total volume of the set of three- dimensional structures 32 and the volume of the hydrogel network 31 is ranging from 5% to 70%, preferably from 10% to 50%, of the volume of the growing chamber 30. Alternatively, when the three-dimensional structures 32 are in the form of pits, the hydrogel network 31 is preferably comprised between the interior faces (13, 23) of the upper and lower surfaces (10, 20). In this embodiment, the volume of the growing chamber is the sum of the volume comprised between the interior faces (13, 23) of the upper and lower surfaces (10, 20) and the volume of the pits. The volume of the hydrogel network 31 is ranging from 1% to 70%, preferably from 5% to 50%, of the volume of the growing chamber 30.

[0085] The set of three-dimensional structures 32 is preferably arranged along a determined pattern 32’ . The pattern 32’ defines the position of the projection of the center of each three-dimensional structure 32 on one of the interior faces (13, 23) as represented by the black dots in figure 5. For example, the distance D between the projections is ranging from 20 pm to 500 pm, preferably from 250 pm to 350 pm. The pattern 32’ may be non-periodic but is preferably periodic. The determined pattern 32’ allows to obtain a controlled structured which is well defined when manufacturing the biochip. This thus allows to define specific features for the flow configuration such as stopping points by a wall, bypassed zone, etc.

[0086] In one embodiment, the pattern 32’ comprises a specific orientation for the three- dimensional structure 32. The specific orientation may comprise an orientation axis O of the three-dimensional structures 32. For example, in the embodiment wherein the three- dimensional structures 32 form a structural network, the orientation axis O is parallel tothe axis subtended by the inlet 1 and outlet 5, i.e., parallel to the direction of the flow represented by the dashed arrows in figure 6A. Preferably, the axis subtended by the inlet 1 and outlet 5 is parallel to the direction of the length of the upper and lower surfaces (10, 20). In the embodiment wherein the three-dimensional structures 32 are in the form of pits, the orientation axis O may be present an angle ranging from 10° to 90° with the axis subtended by the inlet 1 and outlet 5, i.e., the direction of the flow.

[0087] In one embodiment, each three-dimensional structure 32 is crescent- shaped or arch-shaped as represented in figures 6A, 6B and 6C. Therefore, each three-dimensional structure 32 comprises a concave surface 32b and a convex surface 32a. Preferably, the concave surface 32b is oriented towards the inlet 1 of the bioship and a convex surface 32b oriented towards the outlet 5 of the bioship. The concave and convex surfaces (32a, 32b) of one three-dimensional structure 32 join together in order to form two tips 32c. The tips 32c are preferably rounded tips. This avoids any damage to the growing cells. Each crescent- shaped three-dimensional structure 32 has a length L measured as the distance separating its tips 32c. Each crescent- shaped three-dimensional structure 32 also has a width W measured as its largest size perpendicular to the direction of the length L. For example, the length L is ranging from 100 pm to 900 pm, preferably from 500 pm to 700 pm. The width W is preferably about 50% of the length L. For example, the width W is ranging from 150 pm to 450 pm, preferably from 250 pm to 350 pm.

[0088] In one embodiment, the determined pattern 32’ forms at least two rows, each row comprising at least two crescent-shaped three-dimensional structures 32. The tips 32c of crescent-shaped structures 32 of each row are preferably aligned in order to form a single straight-line R, i.e., the rows are substantially perpendicular to the direction of the flow represented by the arrows in figure 6A. Alternatively, the tips 32c of crescent-shaped structures 32 of each row are aligned in order to form two straight lines R so that the rows are substantially parallel to the direction of the flow represented by the arrows in figure 6B.

[0089] The three-dimensional structures 32 may be formed by molding or by additive manufacturing such as 3D printing, preferably at the same time as the surface (10, 20) on which they are disposed.

[0090] Figure 3 represents an embodiment wherein the biochip 100 comprises an intermediate layer 40 disposed in the growing chamber 30. Preferably, the intermediate layer 40 is disposed between the upper surface 10 and the lower surface 20. The position of the intermediate layer 40 thus corresponds to the interfacing plan I. The intermediate layer 40 is characterized by a thickness which is small compared to the thickness of the upper surface 10 or the lower surface 20. For example, the intermediate layer 40 has a thickness ranging from 0.1% to 10% of the thickness of the upper surface 10, preferably from 2% to 5%.

[0091] In one embodiment, the intermediate layer 40 is disposed on the upper or lower surface (10, 20) as represented in figure 3. In this embodiment, the intermediate layer 40 is in contact and covers the surface onto which it is disposed. If the inlet 1 and / or the outlet 5 are disposed on the surface onto which the intermediate layer 40 is disposed, the intermediate layer 40 may be porous in order to let the fluid pass from the inlet 1 and / or the outlet 5 to the growing chamber 30 through the intermediate layer 40. In another example, a second set of inlet 1 and outlet 5 may be disposed on the surface (10, 20) opposite the intermediate layer 40. With two sets of inlets (1) and outlets (5), it is possible to impose a first flow inside the growing chamber 30 - typically a culture medium - and a second flow with different composition and / or flow rate - typically a chemical of interest interacting with the cells, either toxic or therapeutic - to mimic a constant medium of interaction. The intermediate layer 40 may be a filter. Alternatively, the intermediate layer 40 may be watertight. Advantageously, the intermediate layer 40 is a transparent polymer, for example a polyester.

[0092] In an alternative embodiment, the intermediate layer 40 is not disposed on the upper or lower surface (10, 20). The intermediate layer 40 thus allows to physically separate the growing chamber 30 into two sub-chambers on either side of the intermediate layer 40. The hydrogel network 31 may be placed in one of the two sub-chambers or in the two sub-chambers. Placing the hydrogel network 31 in the two sub-chambersadvantageously allows to perform two different cell cultures, i.e., a co-culture, in the two sub-chambers. The two hydrogel networks 31 may have different chemical, physical and / or mechanical properties to increase the efficiency of both cell cultures. In the embodiment wherein the hydrogel network 31 is placed in one of the two sub-chambers, the intermediate layer 40 may be configured to receive a cell culture allowing to coculture different type of cells. Indeed, for example, a first type of cell may be cultured on the hydrogel network 31 disposed in one of the two sub-chambers while a second type of cell may be cultured on the intermediate layer 40 on the other sub-chamber. Advantageously, the intermediate layer 40 may allow the exchange of fluid and bioactive molecules between the two sub-chambers. The intermediate layer 40 may be functionalized with, for example, proteins. The functionalization may be advantageous to improve the adhesive behavior for the cultured cells. The intermediate layer 40 may be a filter. When the intermediate layer 40 is not disposed on the upper or lower surface (10, 20), the biochip preferably comprises two sets of inlet 1 and outlet 5. The first set of inlet 1 and outlet 5 are disposed on the upper support 10 while the second set of inlet 1 and outlet 5 are disposed on the lower support 20.

[0093] The difference in the height of the sub-chambers is advantageous. Indeed, the sub-chamber with a larger volume is able to comprise a larger hydrogel network 31 and thus a larger quantity of cells whereas the sub-chamber with a lower volume is able to contain a lower quantity of fluid which reduces the cost of using the biochip 100 especially in case of expensive nutritive fluids. Alternatively, the intermediate layer 40 may be a filter having a molecular weight cut-off configured to retain the more valued molecules in the sub-chamber comprising the cells, while, on the other sub-chamber, a less expensive fluid comprising the nutrient medium is perfused. For example, the molecular weight cut-off may be configured to allow glucose diffusion through the intermediate layer 40, while retaining growth factors.

[0094] The intermediate layer 40 may be an adhesive layer used to bind the upper surface 10 and the lower surface 20 in a reversible or sustainable manner.

[0095] In the embodiment wherein the intermediate layer 40 is not an adhesive layer, the upper and lower surfaces (10, 20) may be bound to the intermediate layer 40 on a bindingarea 45. The binding area 45 represents preferably less than 5% of the interior face (13, 23). This low binding area advantageously allows the reduction of costs when manufacturing the biochip. Moreover, it allows an easier destruction of the biochip in order to recover the three-dimensional structures 32 for biological, mechanical and / or chemical analysis.

[0096] The binding area 45 is preferably on the periphery of the biochip 100. This is advantageous because it allows to avoid any contamination to the cells in the growing chamber 30.

[0097] The biochip 100 may further comprise a spacer 50 disposed between the upper surface 10 and the lower surface 20. The spacer 50 is illustratively represented in figure 4. Contrarily to the intermediate layer 40, the spacer 50 is characterized by a thickness which is of the same the same order of magnitude as the thickness of the upper surface 10 or the lower surface 20. For example, the spacer 50 has a thickness ranging from 50% to 150% of the thickness of the upper surface 10. The presence of a spacer 50 is advantageous because grooving the upper surface 10 and the lower surface 20 is not necessary to form the growing chamber 30. Indeed, the spacer 50 comprises an opening through its thickness. Therefore, the periphery of the opening allows to space out the upper surface 10 and the lower surface 20, the opening thereby creating a volume between the upper surface 10 and the lower surface 20 and forming the growing chamber 30. In other words, the spacer 50 forms the walls (or border) of the growing chamber 30, the upper surface 10 and the lower surface 20 closing the growing chamber 30 from the top and the bottom. The spacer 50 is further advantageous because it allows to combine several physical characteristics according to the different material used for the upper and lower surfaces (10, 20) and for the spacer 50. For example, the spacer 50 may have sealing properties allowing to avoid any leakage when the upper surface 10 and the lower surface 20 are abutted.

[0098] The intermediate layer 40, if present, may disposed between the spacer 50 and the upper surface 10 and / or between the spacer 50 and the lower surface 20.

[0099] The biochip 100 may further comprise a coating 55 on at least one of the surfaces of the growing chamber 30. Contrarily to the surfaces (10, 20), the intermediate layer 40 and the spacer 50, the coating 55 is a thin layer deposited on a surface. In other words, it is not a supplementary element which increases the total height of the biochip.

[0100] When the coating 55 is disposed after the assembly of the upper and lower surfaces (10, 20) of the biochip and formation of the hydrogel network, z.e., when the hydrogel network 31 is present, the coating 55 is disposed on the overall surfaces of the growing chamber: the hydrogel network 31 , the interior faces (13, 23) of the upper and / or lower surfaces (10, 20), if not covered by the hydrogel network 31 the intermediate layer 40 if present, and the spacer 50 if present.In this embodiment, the coating 55 is preferably hydrophilic so as not to reduce the nesting efficiency of the cells on the hydrogel network 31. Different coatings 55 may be deposited in the growing chamber 30 allowing to culture different type of cells.

[0101] It appears clearly that several coatings 55 may be applied during the preparation of the biochip 100, leading to different adhesion properties for cells under culture. For instance, upper and lower surfaces (10, 20) may be coated partially to improve hydrogel wetting in order to obtain a growing chamber completely covered by hydrogel material in some areas, and with naked plastic surface in other areas, the latter being coated with another treatment specific to plastic. Such a configuration could lead to co-culture of two type of cells being adhesive on the hydrogel network for a first type of cell and on plastic for the second type of cell.

[0102] The coating 55 may be a fluorinated polymer. The fluorinated polymer advantageously prevents adsorption of molecules of biological interest such as proteins and / or antibodies. For example, the coating 55 may be a film of perfluoropoly ether. The coating 55 may comprise proteins alone or in mixtures with an extracellular matrix (for example, collagen, Matrigel™, fibronectin, vimentin etc.), at least one specific antibody recognizing cell membrane sites (for example, CD144, CD31 for endothelial cells; KRT7for cholangiocytes, ALB for hepatocytes etc.), at least one cell-cell adhesion protein such as Cadherin 1.

[0103] Advantageously, thanks to the properties that may be provided with the hydrogel network 31, the coating 55 is not necessarily applied on the hydrogel network 31. The coating 55 may be thus only be disposed before the assembling of the upper and lower surfaces (10, 20) of the biochip thereby simplifying the manufacturing of the biochip 100.

[0104] The biochip 100 of the invention is thus advantageous compared to the known biochips molded in one piece because it allows to insert the spacer 50, the intermediate layers 40 and / or the coating 55 between the upper and lower surfaces (10, 20) in order to improve the degree of freedom of the technical, mechanical and physical properties that may be provided to the biochip 100 thanks to the additional elements between the upper and lower surfaces (10, 20).EXAMPLE

[0105] The present invention is further illustrated by the following example.

[0106] The three-dimensional development of the cells is compared between a biochip comprising a set of three-dimensional structures 32 and a biochip 100 of the invention comprising the same set of three-dimensional structures and a hydrogel network 31. The hydrogel network 31 comprises a cryogel obtained from a formulation comprising a mass fraction of 1% of alginate with 0.12% of adipic acid dihydrazide (AAD) and 0.27% of 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0107] 500 000 Epithelial-like HepG2 / C3A cells 500 are seeded in each biochip and cultured for 6 days with a 10 mL / min perfusion.

[0108] The cell proliferation after 6 days is shown in figure 8 for the biochip 100 of the invention. In the biochip 100, the cells 500 have grown all along the hydrogel network 31. The cultured cells 500 thus occupy the whole height of the growing chamber 30, z.e., up to 200 pm. The density of cells 500 is thus lower with the hydrogel network 31 therefore allowing a better culturing environment for each cell.

[0109] The cells proliferation after 6 days is shown in figure 9 for the biochip without hydrogel network. In this biochip, the cells 500 have preferably grown on the lower surface 20 between the three-dimensional structures 32. The cells 500 form a superposition of layers. The total height of the layers of cells 500 at day 6 reaches 100 pm.

[0110] The biochip 100 of the invention thus allows a three-dimensional cell culture taking the advantage of the whole height of the growing chamber and mimicking cell growth in or on a real organ structure.NUMERICAL REFERENCES1: inlet / / 5: outlet / / 10: upper surface / / l l: exterior surface of the upper surface / / 13: interior face of the upper surface / / 20: lower surface / / 21: exterior surface of the lower surface / / 23: interior face of the lower surface / / 30: growing chamber / / 31: hydrogel network / / 32: three-dimensional structure / / 32’ : pattern / / 32a: convex surface of the three-dimensional structure / / 32b: concave surface of the three-dimensional structure / / 32c: tips of the crescent- shaped three-dimensional structure / / 35: connecting network / / 40: intermediate layers / / 45: binding area / / 50: spacer / / 55: coating / / 100: biochip for cell culture / / 500 - Cultured cells / / A: perpendicular axis / / Bl: border of the upper surface / / B2: border of the lower surface / / D: distance between the centers / / I: interfacing plan / / L: length of the three-dimensional structure / / O: orientation axis / / P: longitudinal plan / / R: line of tips of the crescent- shaped three-dimensional structure / / W: width of the three-dimensional structure

Claims

CLAIMS1. A biochip (100) for cell culture comprising an upper surface (10), a lower surface (20), at least one inlet (1) and at least one outlet (5), each inlet (1) and each outlet (5) being disposed in the upper surface (10) and / or the lower surface (20), wherein the upper surface (10) and the lower surface (20) are configured in order to form a growing chamber (30) between the upper surface (10) and the lower surface (20) when the upper surface (10) is abutted on the lower surface (20); wherein the growing chamber (30) allows fluidic communication from the at least one inlet (1) to the at least one outlet (5); wherein the growing chamber (30) comprises a hydrogel network (31); and wherein the growing chamber (30) comprises at least one of the following configuration elements: a set of three-dimensional structures (32) disposed on the upper surface (10) and / or the lower surface (20); an intermediate layer (40) disposed between the upper surface (10) and the lower surface (20); or a spacer (50) disposed between the upper surface (10) and the lower surface (20), the spacer (50) forming walls of the growing chamber (30), the upper surface (10) and the lower surface (20) closing the growing chamber (30).

2. The biochip (100) according to claim 1, wherein the growing chamber comprises the set of three-dimensional structures (32).

3. The biochip (100) according to claim 2, wherein the set of three-dimensional structures (32) forms a structural network between the at least one inlet (1) and the at least one outlet (5).

4. The biochip (100) according to claim 2 or 3, wherein a total volume of three- dimensional structures (32) computed as a sum of a volume of each three-dimensional structure (32) of the set of three-dimensional structures is ranging from 5% to 50% of the volume of the growing chamber (30).

5. The biochip (100) according to any one of claims 2 to 4, wherein each three- dimensional structure (32) is a pit or a channel in the upper surface (10) and / or the lower surface (20).

6. The biochip (100) according to any one of claims 2 to 4, wherein each three- dimensional structure (32) is crescent- shaped, each three-dimensional structure (32) comprising a concave surface (32b) oriented towards the at least one inlet (1) of the bioship (100) and a convex surface (32a) oriented towards the at least one outlet (5) of the bioship (100), the set of three-dimensional structures (32) being arranged along a determined pattern (32’).

7. The biochip (100) according to claim 6, wherein each crescent- shaped three- dimensional structure (32) has a length (L) ranging from 100 pm to 900 pm and a width (W) ranging from 150 pm to 450 pm, preferably each crescent- shaped three- dimensional structure (32) ends with two rounded tips (32c).

8. The biochip (100) according to claim 6 or 7, wherein the determined pattern (32’) forms at least two rows, each row comprising at least two crescent-shaped three- dimensional structures (32).

9. The biochip (100) according to claim 8, wherein a distance between the centers of two successive crescent- shaped structures (32) of the same row is ranging from 20 pm to 500 pm.

10. The biochip (100) according to any one of claims 6 to 9, wherein each three- dimensional structure (32) is in contact with the upper surface (10) and the lower surface (20).

11. The biochip (100) according to any one of claims 1 to 10, wherein the growing chamber comprises the intermediate layer (40).

12. The biochip (100) according to claim 11, wherein the upper surface (10), the lower surface (20) and the intermediate layer (40) are bound together on a binding area (45) representing less than 5% of an area of the upper surface (10), preferably the binding area is on the periphery of the biochip (100).

13. The biochip (100) according to any one of claims 1 to 12, further comprising the spacer (50).

14. The biochip (100) according to any one of claims 1 to 13, wherein the growing chamber comprises a coating (55) on at least one of the surfaces of the growing chamber (30).

15. Manufacturing process of the biochip (100) according to any one of claims 1 to 14, the process comprising:Injecting a polymer formulation in the growing chamber (30);Forming the hydrogel network (31) in three dimensions from the polymer formulation.

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