Cell culture device

Abstract

The invention relates to a cell culture device comprising a receptacle (2) for cells, which receptacle rests on a base (10). The receptacle (2) is produced in a single piece and has a first face (30) which rests on the base (10), a second face (40) which substantially faces the first face (30), and a cavity (9) having an opening (8) at the second face (40) and a bottom (50) close to the first face (30). The cavity (9) comprises at least one groove (4) which is arranged in the bottom (50) and which forms a culture portion arranged to receive cells (7) and to enable the culture thereof, and a guide (60) which comprises at least one inclined surface (3) which connects the second face (40) to the groove (4). The inclined surface (3) has a surface state for guiding cells, deposited on the second face (40) substantially at the groove (4), into said culture portion by gravity, in the manner of a funnel.

Description

Description Title of the invention: Cell culture device

[0001] The present invention relates to a three-dimensional cell culture device enabling self-organization of cells in the form of a three-dimensional circular ring.

[0002] In vitro procedures using cells are increasingly present in research, particularly in the initial phases of research for new molecules or drugs (preclinical tests). Their use is essential to avoid massive recourse to animal tests (in vivo tests), especially since the use of such techniques also offers numerous advantages such as ease of use, low costs, and their rapid implementation.

[0003] The critical aspect of these in vitro procedures is the cells and their response to the molecules being tested, drugs, or external stimuli. Although very useful, most of the procedures used today rely on the use of two-dimensional cell monolayers. In these situations, cells are grown on flat, rigid substrates, such as multi-well cell culture plates (such as ELISA plates). When comparing the 2-D in vitro environment of cells with the in vivo environment, it is concluded that the results obtained do not correspond to what actually happens in the human body. These unsuitable cell culture conditions are probably the cause of the low success rate in clinical testing of new drugs.

[0004] This is why there is a current need for in vitro models that accurately mimic the natural environment of cells, thus enabling reliable drug screening. To avoid the drawbacks of existing models, in vitro testing procedures have been developed to reproduce a three-dimensional environment that is more faithful to the in vivo microenvironment of cells, with more precise control of physical and biochemical properties, allowing the cells tested to respond in a manner closer to reality.

[0005] Spheroids are a promising three-dimensional cell culture model for reflecting the in vivo environment of cells. These multicellular aggregates are unorganized self-assemblies of cells. For several years, tumor cell spheroids have been considered capable of approximating the complexity of tumors in vitro and being used for the characterization of new treatments. However, these structures have several disadvantages. First, spheroids are difficult to manipulate and characterize without disrupting their self-assembly. It is difficult to acquire images of cells at the interior of the spheroid without manipulating it. Furthermore, for many organs, the natural organization of the cells of this organ in a simple spheroid does not allow reproducing the organization observed in vivo. The cells at the heart of the spheroid also experience an oxygen deficit which may not be desirable for the intended experiment.

[0006] EP 3425043 describes a screening method using spheroids organized in a multi-well culture plate. The spheroids are formed in U-shaped wells whose walls are non-adherent for the cells. The effect of the tested drugs is evaluated by validating whether a spheroid is self-assembled or not.

[0007] Despite the compatibility of this method with the multi-well plates commonly used in screening platforms, it has several drawbacks. The U-shape of the wells degrades the quality of spheroid imaging. In addition, the response obtained (the effect of the molecules) is only qualitatively assessed by the formation or absence of spheroid formation. Another disadvantage of this type of device is that it does not allow the organoid to be held in place, making it difficult to change the surrounding culture medium without moving the spheroid.

[0008] Another important aspect of in vitro cell culture models is the parameters that can be measured and the ease with which these measurements are performed. In addition, the adaptability of these procedures to large-scale rapid drug screening platforms is of great importance.

[0009] Patent application US2017-0089887 describes an in vitro model for measuring cell contractility. Cells are first magnetized (by adding magnetic nanobeads) and placed in wells above ring-shaped magnetic fields that impose a ring organization on the cells. The magnetic field is removed after 3D self-assembly of the cells. Test compounds are added to the wells and the acquired images are used to quantify the contractility of these cell rings. The images are compared before and after the addition of the test compounds.

[0010] While the contractility or relaxation of the ring can be observed, this method does not allow the force exerted by the cells to be measured. Furthermore, the cells must be modified (adding magnetic beads) before being cultured.

[0011] WO 2017-008699 entitled "Cell traction force measuring device, measuring method and preparation method" describes a measuring device using a layer of nano-pillars of known Young's modulus on top of which cells are cultured. Analysis of the images reveals the deformation of the nano-pillars, and thus a map of the forces applied by each cell can be established. This method is compatible with high-resolution imaging, making it possible to understand what happens at the cellular and subcellular level. However, this method measures the forces exerted by individual cells that are not necessarily representative of the pathology and / or organ that we wish to study. In addition, the fabrication of nano-pillars is expensive and complex. This prevents the adaptation of these methods to multi-well plates that are standard for drug screening.

[0012] US 9250241 describes the use of deformable substrates coated with micropatterns, which are themselves coated with fluorescent adherent proteins and are surrounded by cell-nonadhesive polymers. Cells are grown on these substrates and are located exclusively in the adhesive areas. Traction is quantified by comparing images of the micropatterns with and without cells on the patterns. Analysis can then be performed by comparing the shape of the fluorescent patterns.

[0013] This micropattern-based method is focused on individual cell measurements. This does not allow for reproducing the natural environment of cells composed of multiple cells that promote their natural communication and organization. Furthermore, this method does not allow for the creation of 3D cell assemblies since the cells adhere to a substrate. The resulting organizations are therefore poorly representative of the 3D organization of cells in vivo.

[0014] A 3D process combining cardiomyocyte cells (derived from induced pluripotent cells) and an extracellular hydrogel matrix derived from decellularized porcine hearts has recently been described in I. Goldfracht et al., Acta Biomaterialia (2019) 145-159. Cardiomyocytes combined with this matrix are arranged in annular molds, demonstrating spontaneous and synchronized contractility that starts within 24 / 48 hours and continues for up to 6 months. Gene expression analysis demonstrated a signal of adequate maturation over this time scale.

[0015] The functional properties of the resulting micro-core were evaluated by studying genetically encoded calcium and fluorescent indicators sensitive to electrical voltage with optical imaging and force measurement techniques.

[0016] The main drawback of this technology is the ease of use of the process. The cardiac tissue rings must be removed from the molds, which is difficult to handle. Furthermore, imaging of individual cells is not possible because the rings are too thick, while ring imaging and strength assessment cannot be performed simultaneously, resulting in a time-consuming and expensive process.

[0017] The way cells organize themselves, the 3D geometry of cells, and the geometry of their microenvironment are important factors from a biological perspective because they can impact cell maturation, physiology, and functional behavior. For example, the speed and nature of cell differentiation Induced pluripotent stem cells may depend on these factors and have an impact on the behavior of the cells and therefore the representativeness of the experimental results. Furthermore, the representative measurement of certain physical quantities such as the contraction force of cells requires that these cells be organized in a similar way to what is found in a living organism, for example in fiber for muscle cells.

[0018] The culture of 3D cells in currently known systems, especially the culture of cells with contractile activity, is limited for the following reasons:

[0019] - Cells can take a long time to organize into 3D structures, and their organization is generally not representative of that observed in vivo. - There are often a large number of cells that are lost during the process - Cells may need to be modified before being cultured (e.g., magnetizing cells with nanobeads) - The produced organoid is not always maintained, which makes it difficult to replace the surrounding environment. - Compatibility of processes with drug screening platforms is not always possible, especially with multi-well plates on which industrial automation of drug screening is commonly based. - Acquiring images with adequate resolution is not always possible and requires manipulation of 3D structures. - The results are limited or difficult to use (qualitative or single cell observations) - Measurement of parameters such as cell contractile force is not always measurable.

[0020] The invention improves the situation. In particular, the invention makes it possible to avoid the disadvantages of currently known 3D systems by providing a three-dimensional cell culture device, preferably made of an optically transparent material such as a hydrogel or a biocompatible polymer material, this device allowing the spontaneous assembly of cells into aggregates.

[0021] Thus, the subject of the invention is a cell culture device comprising a receptacle for cells which rests on a base, the receptacle being made in one piece and having a first face which rests on the base and a second face substantially opposite the first face, as well as a cavity having an opening at the level of the second face and a bottom close to the first face, which cavity comprises at least one groove arranged in the bottom and forming a culture portion arranged to receive cells and allow their culture, and a guide which comprises at least one inclined plane which connects the second face to the groove, which inclined plane has a surface state arranged to guide cells deposited on the second face substantially at the level of the groove in said culture portion by gravity in the manner of a funnel.

[0022] In particular embodiments:

[0023] - the inclined plane can surround the groove; - the groove can be arranged in a closed path in the cavity; - the groove may be arranged in an annular shape in the cavity; - the device may further comprise a central pillar arranged in the center of the groove; - the central pillar may be surrounded by the groove and at least part of the perimeter of said central pillar may define the internal perimeter of the groove; - the central pillar can be arranged in a general S-shape (i.e. have a circumference that narrows below a certain height); - the device can be made at least partially, preferably completely, from a transparent material; - the material of the device may have a refractive index of between 1.32 and 1.45, preferably equal to 1.33, so as to allow the acquisition of in situ images of individual cells or of an aggregate of cells; - the device may be made at least partially, preferably totally, from a flexible or soft biocompatible hydrogel-based material making it possible to measure the contraction force of the aggregate of cells forming the annular-shaped tissue; and - the surface roughness Ra of the device can be between 0.2 and 0.3 pm.

[0024] In general terms, the cell culture device described herein is three-dimensional. It has a structured substrate, comprising a hollow cavity having a shape that can be described as a crater, the bottom of which has a groove in the form of a path in which the cells can spontaneously organize themselves into a continuous three-dimensional tissue of shape following the path. The path can be of any shape, provided that the groove it defines has an aspect ratio (length of the groove path divided by the width of the groove) greater than or equal to 2, which will lead to the formation of an aggregate of cells in the shape of the groove defined by the path.

[0025] The path can be open (straight or irregular line, a network, a grid, a tree shape) to mimic the organization of elongated organs such as vessels, or glandular organs.

[0026] Preferably, the path may be closed (ring, polygon, or other closed irregular path) if the cells are contractile. To facilitate analysis, the path may advantageously take the form of a ring forming a ring-shaped groove in which the cells can spontaneously organize into a continuous three-dimensional ring-shaped tissue.

[0027] The groove is flanked on at least one of its sides and along the entire path by inclined flanks whose slope points towards the groove and whose width is at least equal to the width of the groove. Preferably, these flanks have an angle greater than 30° relative to the horizontal. Preferably, the flanks are of the same width around the entire circumference of the path.

[0028] When cells are seeded on top of this substrate, the cells fall to the bottom of the substrate under gravity or an artificially applied force such as centrifugal force so that they are guided along the flanks towards the groove, leading to the formation of a cell aggregate or tissue whose shape matches that of the groove.

[0029] The structured culture substrate is preferably composed of a flat support on which the grooves in which the cells spontaneously organize themselves into a tissue have been formed (preferably molded).

[0030] According to a preferred embodiment, the opening of the cavity is circular and has a diameter greater than the diameter of the annular groove.

[0031] Preferably, the walls of the cavity completely surround the annular groove.

[0032] According to a preferred embodiment, the device has a central pillar arranged in the center of the groove when the latter is annular in shape.

[0033] According to an alternative embodiment, the central pillar is completely surrounded by the annular groove and preferably delimits the internal perimeter of the annular groove. Advantageously, the central pillar has a local reduction of its perimeter to an intermediate height in order to allow reliable and constant maintenance of the position of the cell ring around the pillar, in particular when the cells contract or if an external force is applied to the cell ring, for example if the culture medium is replaced. This local reduction of the perimeter prevents the ring from sliding along the pillar if it contracts around it.

[0034] According to a preferred method, the local reduction in perimeter is less than 20% of the average perimeter of the central pillar to facilitate the demolding of the structure.

[0035] Thus, the central pillar can take a general s-shape. This s-shape allows for reliable and constant maintenance of the position of the cell ring around the pillar, particularly when the cells contract. As explained above, this local reduction in the perimeter prevents the ring from sliding along the pillar, advantageously Fortunately, maintaining the ring allows for reliable and reproducible observations and measurements to be made over time.

[0036] According to a preferred embodiment, the local reduction in perimeter is less than 20% of the average perimeter of the central pillar to facilitate the demolding of the structure. According to a preferred embodiment, the device according to the invention is characterized in that the base of the central pillar is integral with the structured substrate and in that the other end of the central pillar is preferably a conical point to guide the seeded cells towards the groove.

[0037] According to another preferred embodiment, the device is made at least partially, preferably totally, of transparent material, preferably with an optical refractive index of between 1.32 and 1.45, preferably equal to 1.33, so as to allow the acquisition of in situ images of individual cells or of an aggregate of cells.

[0038] According to a preferred embodiment, the device is made at least partially, preferably totally, of a flexible or soft material making it possible to measure the contraction force of the aggregate of cells forming the annular tissue. The behavior of the central pillar can be compared to an elastic solid. Thus the contraction force, (F), exerted by the 3D cellular structure is proportional to the variation in the radius of the pillar (A r) multiplied by a constant (a) which depends on the mechanical properties of the material, such as its modulus of elasticity or Young's modulus (F "and A r).

[0039] Advantageously, the Young's modulus of the material is in the range of 500 Pa to 100 kPa and of the same order of magnitude as the pressure exerted by the ring expressed approximately by the expression P=T / (Rh) where T is the contractile force exerted by the cells along the ring (in Newton), R is the radius of the pillar at the point where the ring encloses it (in meters) and h is the contact height between the ring and the pillar (in meters)

[0040] Preferably the flexible or soft material is a biocompatible material or a hydrogel allowing the diffusion of nutritional elements and / or oxygen and / or allowing the measurement of the contraction force of the ring-shaped cell aggregate.

[0041] According to an alternative embodiment, the device is made partially or totally from a photo-polymerizable polymer material in order to reduce its manufacturing time.

[0042] To allow the cells to concentrate in the ring-shaped groove, the walls of the cavity are made of cell-non-adherent material to allow the cells to freely exert their own force on the aggregate.

[0043] According to another preferred embodiment, the lower part of the groove (bottom), to which the cells adhere, can be adherent to provide support for the formation of the aggregate, which makes it possible to stabilize its shape and prevent the aggregate from The cell contracts on itself and takes the form of a simple spheroid.

[0044] Compared to prior art devices, the device according to the invention makes it possible to produce stable ring-shaped organoids after a single cell seeding step.

[0045] The invention also relates to a multi-well support, which comprises a plurality of cell culture devices according to one of the forms and / or variants indicated in the present application, preferably distributed regularly on the surface of the support.

[0046] According to another aspect, the invention also relates to a method of manufacturing a device as described above, characterized in that it comprises in particular the following steps:

[0047] - Prepare a substrate preferably made of a transparent material (e.g. glass or plastic, e.g. in Petri dish format, multi-well plate or glass slide) - Deposit a layer of an unpolymerized monomer solution on the substrate - Create an impression using a mold in the unpolymerized layer having the shape of the device according to one of the variants described above so as to create a structured substrate - Polymerize the monomer solution of said layer so as to harden the structured substrate - Demould the hardened structured substrate.

[0048] Finally, the culture device according to the invention, the multi-well support and the method can be used for carrying out cell culture, in particular cells derived from human cells.

[0049] Among the many advantages of the invention, it will be noted that the device of the invention makes it possible to maintain autonomous organoids on a structured substrate while allowing flows in the culture medium above and on the annular groove without the need to use any adhesive between the cells and the substrate to avoid disturbing the cells.

[0050] The operation of the device according to the invention makes it possible to optimize the number of cells required to form the three-dimensional ring structure. Few cells are lost because the vast majority of them are guided towards the ring formation zone (groove) due to the presence of the sides (preferably inclined) of the cavity which ensure the guidance and sliding of the cells in the groove. Such a device therefore has a structure which generates a robust and reproducible technique for assembling cells into 3D structures.

[0051] The invention can also be used to measure contractility forces when the material constituting the structured substrate is flexible or soft. The measurement can be performed by analyzing the deformation of the central pillar (contraction) created by the contraction of the 3D cell ring or organoid. This allows for example to measure the contractility force of contractile cells such as striated muscle cells (skeletal muscle cells or cardiac cells), smooth muscle cells (pericytes, vascular smooth muscle cells, visceral or bladder smooth muscle cells, etc....), mesenchymal contractile cells (fibroblasts) or any other cells with contractile behavior and also to measure changes due to the effect of specific compounds, chemical cell environment or cell modification.

[0052] In addition to the internal contractility of the fiber formed by the cell ring, the invention can also be used to measure the contractility induced by its surface tension of the cell aggregate, in particular for non-contractile cells. For example, to indirectly measure the surface tension of an epithelial cell aggregate.

[0053] To measure the contraction force of cells in the organoid, the diameter variation measurement is used to assess the variation in contraction force applied by the cellular structure on the central pillar.

[0054] For small deformations, dimensional analysis shows us that the force applied by the organoid can be determined as follows:

[0055] dF = dO.E.dD

[0056] where dF is the change in force in Newton, dD the change in the measured diameter of the central pillar, E the modulus of elasticity of the pillar in N / m2 and dO a characteristic length which depends on the precise geometry of the central pillar.

[0057] Advantageously, an active ingredient or drug such as Latrunculin A can be used to inhibit cell contraction and thus deduce the contraction force before application of the drug.

[0058] The device can also be used to measure contraction force profiles over time by making movies or time-lapses of the organoid. In this case, the device can be used to measure the contraction profile of muscle fibers, spontaneous or under the effect of a stimulus, in the presence or absence of the molecules whose effect is to be measured. For example, to study the effect of molecules acting on the contractility of cardiac muscle, skeletal muscles or smooth muscles.

[0059] Another use of the device according to the invention is to produce 3D cell aggregates like spheroids, but having a higher surface / volume ratio which have the advantage of improving the diffusion of nutrients and oxygen in the organoids compared to spheroids.

[0060] Yet another use of the device according to the invention consists in making 3D cell aggregates in a single seeding step of cells that remain stuck on the device, due to the contraction of the ring-shaped organoid around the pillar it surrounds. This allows the culture medium to be replaced around the organoid without disturbing it.

[0061] The structured substrate of the device according to the invention can be manufactured using any type of material which has the ability to polymerize and which is biocompatible.

[0062] Monomer molecules react to form a three-dimensional network or polymer chain through polymerization and forming polymers which can be synthetic or natural in nature.

[0063] Preferably, the polymer will be a hydrogel allowing the diffusion of nutritional molecules and the oxygenation of the organoid and therefore its well-being.

[0064] Below are a number of monomers used to generate polymer materials (gels and especially hydrogels)

[0065] -Poly (2-Hydroxyethyl methacrylate)- PHEMA

[0066] -2-Hydroxyethyl methacrylate - HEMA

[0067] -Polyethylene glycol (PEG)

[0068] -Methacrylic acid (MA A)

[0069] -Acrylamide / acrylic acid copolymer

[0070] -Poly(N-isopropylacrylamide)

[0071] -Peptide-based polymers that are not hydrogels can also be used, or elastomers such as silicones (e.g., polydimethyl siloxane) with which it is easy to mold such shapes.

[0072] Preferably this polymer will be photo-polymerizable to speed up the manufacturing process.

[0073] Ideally, the polymer will be optically transparent (refractive index close to that of water 1.33) after polymerization, which allows optical microscopic imaging (e.g. by fluorescence, by high-resolution imaging, ...). Results can thus be obtained based on image analysis.

[0074] The stiffness of the material can be adjusted to suit the cell environment. Therefore, materials with a stiffness between 0.1 KPa and 1 MPa, preferably between 1 and 20 KPa, should be chosen (or adapted).

[0075] The material constituting the structured substrate 1 (at least the walls of the cavity) will preferably be a non-adherent material to help guide the cells towards the annular groove and / or to prevent adhesion of the organoid to the substrate. In cases where adhesion is desired, a material allowing adhesion of the cells will be used. This can be further improved by the presence on the surface of a specific extracellular matrix such as collagen, fibronectin, etc.

[0076] The dimensions of the annular groove can be adapted to the size of the organoids used in the process within the limits described above.

[0077] The invention will be better understood with the aid of the following exemplary embodiments in conjunction with the figures which represent:

[0078] [Fig.l] shows a sectional view of a device according to the invention;

[0079] [Fig.2] shows a top view of a device according to the invention;

[0080] [Fig.3] shows a sectional view of a device according to the invention with respective dimensions;

[0081] [Fig.4] shows steps of molding a device according to the invention;

[0082] [Fig.5] shows photographs of molds for the manufacture of the device according to the invention;

[0083] [Fig.6] shows a photograph of fibroblast cells after centrifugation;

[0084] [Fig.7] shows a photograph of the cells of [Fig.6] one hour after centrifugation and polymerization of the collagen;

[0085] [Fig.8] shows a sectional view of an alternative embodiment of the device according to the invention;

[0086] [Fig.9] shows a sectional view of a variant of the device according to the invention with respective dimensions;

[0087] [Fig.10] shows a photograph of MEFs cells 22 hours after culture in the ring and 12 days after; and

[0088] [Fig.11] shows a diagram of the contraction and relaxation of a central pillar caused by tension-bearing cells arranged around the pillar.

[0089] The figures, tables and description below contain, for the most part, elements of a certain nature. The figures and tables are an integral part of the description, and may therefore not only serve to better understand the present invention, but also contribute to its definition, where appropriate.

[0090] The invention is now described with reference to Figures 1 to 11. In the figures, the same elements bear the same references.

[0091] [Fig. 1] shows a vertical sectional view of the structured cell culture substrate 1 according to the invention. The substrate comprises a receptacle 2 which rests on a base 10. The substrate further comprises a groove 4 having an annular shape. In order to give this annular shape to the groove 4, a central pillar 6 is arranged surrounded by the groove 4, this central pillar 6 having a top 5 of preferably conical shape. Above the annular groove 4 is arranged a wall structure having the shape of a funnel (preferably conical). More particularly, the receptacle is made in one piece (or monobloc) and has a first face 30 resting on the base 10. The receptacle further comprises a second face 40 substantially opposite the first face 30. The entire structure of walls and the annular groove 4 form a cavity 9 having an opening 8 at the level of the second face 40. The funnel-shaped wall structure of the cavity 9 connects the inner edge of the opening 8 located at the top of the cavity 9 to the outer rim of the annular groove 4 located at the base of the cavity 9. More particularly, the cavity 9 comprises the groove 4 which is arranged in a bottom 50 of the cavity. The groove 4 forms a culture portion arranged to receive cells 7 and allow their culture. For this, the cavity 9 further comprises a guide 60 which comprises at least one inclined plane 3. The inclined plane 3 connects the second face 40 to the groove 4. According to the invention, the inclined plane 3 has a surface state arranged to guide cells deposited on the second face 40 substantially up to the level of the groove 4. The cells thus migrate into the culture portion by gravity in the manner of a funnel.

[0092] When cells are arranged on the structured substrate 1, or more precisely on the second face at a guide 60, the funnel-shaped wall structure guides the cells towards the annular groove 4, which leads to the spontaneous formation of a three-dimensional aggregate of annular cells. For this, the guide 60 comprises an inclined plane 3. If necessary, the conical tip of the central pillar 5 helps the cells to slide downwards towards the groove 4 and prevents them from catching on the wall structure, especially when the latter is made of a material with a low coefficient of friction. In this preferred embodiment, the tip 5 is conical and preferably has an apex angle equal to or substantially equal to the angle at the apex of the funnel.

[0093] In [Fig.1], we can see the 7 cells schematically represented which partially overlap to form the annular aggregate.

[0094] The dimensions of the annular groove may vary within wide limits in order to facilitate the formation of cell aggregates according to the invention. Referring to [Fig. 3], the depth hl of the annular groove may vary between 0 and 3000 microns, preferably between 50 and 200 microns, the outer diameter of the groove dl between 10 and 10000 microns, preferably between 200 and 700 microns, the width of the annular groove W 1 between 3 and 3000 microns, preferably between 50 and 200 microns.

[0095] The cavity 9 preferably having a conical funnel shape along its walls will have a height h2 between the opening 8 and the outer edge of the ring of between 0 and 2000 microns, preferably between 100 and 300 microns. When h2 is equal to 0 the device has a configuration limited to the annular groove which allows the formation of the cell ring, but without directing the cells towards the groove 4, which reduces the number of cells in the device. The apex angle Alpha 1 can vary between 0 and 90 degrees, preferably between 40 and 50 degrees so as to allow the cells introduced into the cavity 9 to slide into the annular groove.

[0096] The central pillar preferably defining a vertical axis of symmetry of the device according to the invention, will have a height h3 of between 1 and 4000 microns, preferably between 150 and 550 microns, a diameter d2 of between 1 and 5000 microns, preferably between 100 and 350 microns, a vertical part of height h4 of between 1 and 3000 microns, preferably between 60 and 150 microns, and an apex angle Alpha 2 of between 0 and 180 degrees, preferably between 80 and 100 degrees, (an angle zero or 180 corresponding to a pillar with a flat top), of height hl.

[0097] The device can also take the form described in Figures 8 and 9. The wall structure of the conical cavity 9 has an inclined plane 13 which ends at the edge of the annular groove 4; the central pillar 6 has two lateral faces 15 extending to the groove 4 which, unlike those of the pillar 6 shown in [Fig.l], are not straight but have an S shape, giving the groove 4 a greater width at its lower part than that of this groove at the base of the pillar 6 (in this figure). Thus this particular shape allows the formation of an organoid which can slide upwards under the action of the cells, while remaining fixed on the central pillar thanks to its geometric shape forming a protuberance 16 in [Fig.8].

[0098] Alpha 3 can vary between 0 and 90 degrees, preferably between 40 and 50 degrees, rl and r2 can vary between 0.01 mm and 0.9 mm, preferably between 0.05 and 0.1 mm.

[0099] Cells can be cultured in two ways: either simply suspended in a cell culture medium, or mixed with an extracellular matrix, either natural or synthetic, such as collagen types I, II, III, IV and V. Collagen is the main component of the natural extracellular matrix in the human body; this protein is present between cells in many connecting tissues. It can also be matrigel, hydrogel or other extracellular matrices.

[0100] The concentration or quantity of cells that should be seeded onto the substrate depends on the cell type and the diameter of the cavity compared to the surface area of ​​the ring.

[0101] The production of the structured substrate can be carried out simply by molding using a mold allowing the desired structure to be printed (see for example below [Fig.4]) in the material used.

[0102] Because this molding process is simple, a plurality of devices can be easily molded in a single step to make multi-well plates.

[0103] [Fig. 4] illustrates a method of manufacturing the device according to the invention. A preferably flat support 23 (Petri dish, glass plate, multi-well plate, etc.) is coated with a layer 24 of the chosen polymer (in the unpolymerized state) in liquid or soft form, on which the micro-structured substrate 1 is printed using a mold 20 having the negative shape of the structured substrate 1. Then the poly- merization starts, and the coated layer 24 takes the desired positive structure shape of the micro-structured substrate 1. After demolding ([Fig.4]) the final 3D micro-structured substrate 1 is obtained.

[0104] Ideally, the mold surface should preferably be very smooth with a roughness of less than one micron. Therefore, molds should preferably be manufactured using high-resolution processes (digital milling, 3D printing using two-photon lithography).

[0105] The device according to the invention allows the use of any type of biological cells that can adhere to each other, such as endothelial cells, cardiac cells, smooth muscle cells, epithelial cells, condrocytes, fibroblasts, etc.

[0106] The structured substrate of the device according to the invention can be manufactured using any type of material which has the ability to polymerize and which is biocompatible.

[0107] Monomer molecules react to form a three-dimensional network or polymer chain through polymerization and forming polymers which can be synthetic or natural in nature.

[0108] Cells can be cultured in two ways: either simply suspended in a cell culture medium, or mixed with an extracellular matrix, either natural or synthetic, such as collagen types I, II, III, IV and V. Collagen is the main component of the natural extracellular matrix in the human body; this protein is present between cells in many connecting tissues. It can also be matrigel, hydrogel or other extracellular matrices.

[0109] The concentration or quantity of cells that should be seeded onto the substrate depends on the cell type and the diameter of the cavity compared to the surface area of ​​the ring.

[0110] [Fig.5] represents a mold allowing a plurality of devices according to the invention to be produced.

[0111] The manufacture of a mold of the invention is however accompanied by numerous technical problems that the Applicant had to overcome.

[0112] Microfabrication or micromachining plays a major role in the manufacture of the device of the invention. In technical fields far removed from the invention, such as the semiconductor industry or microfluidic research, miniaturization has seen technological advances in recent years. However, the transposition of this miniaturization into the field of the invention is generally infeasible or even incompatible.

[0113] The Applicant thus first turned to photolithography which is a A technique used to transfer a certain shape or pattern onto a material or surface by selectively exposing light-sensitive polymers using a photomask. Dry etching or wet etching methods can then be used to remove the material from previously selected areas.

[0114] Wet etching is a chemical procedure that is relatively simple and quick to implement. Material can be etched isotropically (orientation-independent) or anisotropically (orientation-dependent). Isotropic etching is the most common. In this method, material is removed uniformly in all directions, which can result in material being removed from under areas that should not be etched. Anisotropic etching (when using crystalline substrates) results in less material being removed under the mask, allowing for better control of wall geometry. Dry etching techniques use ion bombardment toward the surface to selectively remove material. This method can be used to fabricate smaller structures than wet etching, resulting in high-aspect-ratio structures with very good surface quality.Both of these techniques are commonly used, but are severely limited to simple designs, such as straight or vertical walls.

[0115] Additionally, there are additive manufacturing techniques. These techniques play a role in the rapid prototyping of complex shapes. Structures are created by printing layer by layer, based on a design generated by computer-aided design (CAD) software. There are special techniques, such as material extrusion, laser sintering, material jetting, etc. All of these techniques are attractive because they do not require special tools or process modifications, so the initial costs can be relatively low. In addition, they can be used to manufacture rather complex shapes and features, but it should be remembered that the surface quality is always affected by the layer-by-layer printing process.Higher-resolution techniques, capable of achieving sub-micrometer features with excellent surface quality (nanoscale) based on two-photon polymerization, challenge the limitations of conventional additive methods. However, they are only suitable for the fabrication of very small structures. These structures are smaller than the dimensions needed to fabricate a mold that maximizes the surface area used in standard cell culture substrates, such as typical 96-well plates (one well in a typical 96-well plate is approximately 6 mm wide). These plates are commonly used by pharmaceutical companies for high-throughput drug screening. Furthermore, this is a technique used primarily at the research level, with very high costs and long manufacturing times. Additive methods are therefore not suitable for manufacturing the molds that will give rise to the cell culture devices described in the present invention. It is difficult with the use of additive methods to produce in a reasonable time structures with a good quality surface finish, therefore with very high resolution, which have a large volume, which is necessary to produce the device according to the invention allowing a large number of organoids to be produced simultaneously.

[0116] Computer Numerical Control (CNC) machining is a subtractive technique. It starts with a block of material, such as plastic or metal, which is sculpted using specific tools such as drills. It has high dimensional accuracy and allows the manufacture of mechanically robust parts, with a high reproducibility rate from very small to very large scales. The minimum size and complexity of the structures are influenced by the size and shape of the drill head (all machined points must be accessible), the adaptability of the stage (degree of freedom - notably, 5 axes), as well as the speed and sensitivity of response or maneuvering. It can also be used as a post-processing tool, when the base has been primarily 3D printed or manufactured in any other way and it must be precisely adjusted.

[0117] All these techniques come from technical fields far removed from the present invention. EXAMPLE OF EMBODIMENT OF THE INVENTION

[0118] The cell culture device described in the present invention is manufactured by molding. This means that the mold has the negative shape of the final cell culture device.

[0119] To select a suitable method for manufacturing the molds of the invention, the Applicant considered numerous parameters and constraints, including in particular:

[0120] - The use of a robust material that can be used multiple times to mold the chosen polymers such as polyacrylamide or polyethylene glycol hydrogels. The material must also withstand harsh cleaning procedures, which may be necessary to remove residues from the molded materials;

[0121] - A material that does not react and / or adheres to the polymers in question;

[0122] - A method that allows manufacturing at the micron scale and is not limited to straight features, but can adapt to complex shapes such as "s" or curved profiles, inclined walls in particular;

[0123] - The need for surfaces with a very smooth finish, necessary both to facilitate demolding and to avoid reproducing a rough surface in the final cell culture device. This is required so that cells are not trapped by micro-roughnesses on the surface of the polymer, but rather guided by the inclined walls towards the cell assembly zone.

[0124] Considering the above, a process based on material subtraction proved, not surprisingly, to be the most suitable for manufacturing a mold such as the one shown in [Fig.5]. The manufacturing process included the following steps in particular:

[0125] - The selection of a stainless steel material (grade 1.4305), in order to meet at least some of the above-mentioned requirements;

[0126] - A two-phase machining, a first phase in which a laser machine from the company KLM Microlaser GmbH - (KLM Microlaser - KLM El) (3-axis ultrashort pulse laser) is used to mill the bulk material around the conical structures (see [Fig.5]), and a second in which a 5-axis Kern Micro CNC machine from the company Kern Microtechnik GmbH - (KERN Microtechnik - MICRO) was used to smooth the surface around the conical structures, as well as to make the cavity that will give rise to the central pillar;

[0127] - Laser machining of material removal, followed by scanning with a micro-drill. Indeed, laser machining does not allow to obtain a satisfactory surface quality (the roughness was higher than the micrometer scale). It is only after scanning with a micro-drill over the entire or almost the entire surface that thin layers of additional material are removed, until the surface is sufficiently smooth, i.e. with a roughness Ra between 0.2 pm and 0.3 pm. The technique implemented allows to obtain edges with a curvature of approximately 0.1 mm.

[0128] The mold of the invention is thus made of stainless steel having a surface having a roughness Ra of between 0.2 and 0.3 pm. It thus makes it possible to obtain a device of the invention having a roughness Ra of between 0.2 and 0.3 pm.

[0129] - The inner cavity is made in two stages: a first stage in which a drill bit with a diameter smaller than that of the central pillar is used to dig a cylindrical hole, and a second stage in which a spherical profile drill bit is used to finely adjust the profile of the walls, which makes it possible to produce "S"-shaped or curved profiles and a pointed conical hole which gives the conical profile of the central pillar.

[0130] High-resolution computer numerical control (CNC) machining is a flexible technique. To implement the above manufacturing process, many details are taken into account and must be kept in mind when manufacturing a cell culture device of the invention.

[0131] The example below uses a polyacrylamide-based substrate.

[0132] -Mold manufacturing:

[0133] The manufacture of the mold to produce the device according to the invention is shown in Figure 5a (sectional view) and Figure 5b (top view). A mold with an internal shape which is the negative of the structured substrate is used to mold a structure as shown in [Fig.l]. The mold is made of stainless steel (grade 1.4305 in the present example) produced by high-resolution digital milling with an average surface roughness Ra of between 0.2 and 0.3 microns (the smoother the internal surface of the mold, the easier it is to demold the devices. In [Fig.5] we will note the negative shape of the molds compared to the shape of the device, the annular groove (in negative) being located above the conical structure of the walls: in the center of the mold of the annular groove we find a deeper cavity corresponding to the central pillar after molding.

[0134] An acrylamide-based hydrogel was then used to mold the devices in a sterile environment.

[0135] -Preparation of the support:

[0136] The starting support consists of 16 mm diameter glass slides which are cleaned and silanized to promote adhesion.

[0137] -Pre-hydrogel solution:

[0138] After coating the pre-hydrogel solution (acrylamide solution), two different mechanisms can be used to trigger (as catalytic agents) the polymerization of this hydrogel:

[0139] -using UV radiation of appropriate wavelength (e.g., 365 nm) that catalyzes the polymerization of acrylamide and bis-acrylamide free radical by transforming Irgacure 2959 molecules into free radicals. The following reagents are used in the formulation of these types of gels: C3H5O Acrylamide 40%; C7H10O2N2 N,N'-Methylenebisacrylamide 2%; Irgacure 2959 (1% v / v) - UV sensitive (wavelength 365 nm);

[0140] -using ammonium persulfate (APS) to induce polymerization (when decomposing it forms free radicals) of acrylamide and bis-acrylamide monomers and using a free radical stabilizer (TEMED) which is a polymerization promoter.

[0141] The following reagents are used in the formulation of these types of gels: C3H5O Acrylamide 40%; C7H10O2N2 N,N'-Methylenebisacrylamide 2%; (NH4)2S2O8 APS (10%); C6H15N2TEMED.

[0142] In this particular case, the polymerization was carried out by UV photopolymerization. The gel formulation was based on an aqueous solution containing 30% of C3H5O- Acrylamide 40% + 12.5% ​​C7H10O2N2 N,N'-Methylenebisacrylamide 2%; 0.5% Irgacure 2959 (1% v / v).

[0143] -Molding (polyacrylamide):

[0144] The treated glass slides are covered with a 15 microliter drop of pre-hydrogel solution.

[0145] The mold is placed above the coverslips, on the drop of pre-hydrogel solution, and directly irradiated with a UV source of wavelength 365 nm (90 mW, 3 minutes). The gel is demolded and then stored in water. The glass coverslips thus prepared are assembled on a multi-well plate.

[0146] The mold used included 23 microstructures allowing the fabrication of 23 cavities with annular grooves, and therefore to carry out several tests in the same well. The width (wl) of the rings is 100 pm and the diameter of the central pillars is 240 pm.

[0147] Cell culture using fibroblasts derived from human abdominal skin cells:

[0148] The study of fibroblast contractility is currently an important subject for understanding inflammation and healing phenomena.

[0149] -Fibroblast cell protocol:

[0150] Fibroblasts derived from human abdominal skin cells were resuspended in a purified type 1 collagen extracellular matrix with a collagen concentration of 160,000 cells / ml. A 15 microliter drop of the cell mixture was placed on the upper surface of the structured substrate previously incubated in cell culture medium. The substrate was then centrifuged for 2 minutes at 100 G. The cells were guided naturally toward the annular groove under the effect of centrifugal acceleration. The collagen mixture then polymerized at 37 degrees for 1 hour, and then cell culture medium was added to the wells. [Fig. 7] is an image of the fibroblast cells after centrifugation, while [Fig. 8] is an image of the same cells after collagen polymerization (1 hour, 37 degrees) showing that the individual fibroblast cells formed a perfect ring around the central pillar.

[0151] This example demonstrates the rapid and spontaneous assembly of a ring-shaped 3D organoid using the device of the invention.

[0152] -MEFs (Mouse Embryonic Fibroblasts) cell protocol:

[0153] Structured hydrogel substrates with an "s"-shaped central pillar were fabricated using stainless steel molds. The ring width (wl) was 70 μm and the diameter of the central pillar was 360 μm. The hydrogel used was based on polyethylene glycol at a concentration of 6.4%, photopolymerized under UV light.

[0154] MEF cells are first dissociated from their substrate using trypsin and then centrifuged. The cell pellet is then resuspended in a new cell culture medium at a concentration of 160,000 cells per ml. 15 microliters of this cell suspension are spiked onto the top of the gel structure. (The structures are incubated in cell culture medium for 15 minutes beforehand). The substrate containing the cells is then incubated at 37°C, 5% CO2. After 22 hours, the ring-shaped organoids are observed by conventional light microscopy. A self-assembled organoid is visible at this stage, as shown in [Fig. 10]. The organoids are kept in culture for 12 days and the medium is changed every other day. After 12 days, the MEF cell aggregate forms a more compact ring-like structure, as shown in [Fig. 10].

[0155] This example demonstrates the rapid self-assembly of ring-shaped organoids. Since no extracellular matrix was used in this case, the cells sedimented themselves onto the ring-shaped cavity and no centrifugation step was necessary. In this case, the funnel-shaped structure helps guide the cells into the ring-shaped cavity.

[0156] This example also demonstrates that the organoid can be preserved in structured substrates for a long time. The "s"-shaped mold plays an important role, as it allows the ring-shaped organoid to remain in place even when the cells contract and could possibly slide along the central pillar, or when the liquid is agitated, for example when changing the medium. Keeping the organoid securely in place allows for stable observation over long periods while also allowing for easy changing of the surrounding medium.

[0157] In another example, we culture skeletal muscle cells of the C2C12 cell line as ring-shaped organoids and measure their contraction force.

[0158] To measure the contraction force of cells in the organoid, we measure the deformation of the elastic central pillar under the effect of the force.

[0159] The measurement of the variation in the diameter of the pillar (in other words the inner diameter of the ring) is used to assess the variation in the contraction force applied by the cellular structure on the central pillar.

[0160] Advantageously, a drug such as Latrunculin A (inducer of actin depolymerization) can be used to inhibit cell contraction and thus deduce the contraction force before application of the drug.

[0161] The gels used in this example follow the same manufacturing protocol as the previous example with MEFs.

[0162] C2C12 cells are dissociated with trypsin and then centrifuged. The cell pellet is then resuspended in new culture medium at a concentration of 500,000 cells per ml. 200 μl of this cell suspension is pipetted onto the top of the gel structure (previously incubated in culture medium for 15 minutes). The plate is then centrifuged and incubated at 37°C, 5% CO2.

[0163] After 6 hours, the structures were imaged and the diameter of the central pillars measured. To assess the force applied by the organoid on the central pillar, the cells were exposed to medium containing Latrunculin A (1:1000). After 30 minutes of incubation, the structures were imaged again and the diameter of the central pillars measured.

[0164] In our example, the diameter of the central pillar increases by about 1.5% in the presence of Latrunculin A compared to the control experiment without the drug. This is demonstrated in [Fig. I l],

[0165] In particular embodiments: the groove has a bottom and two sides; the groove is arranged in a rectilinear, open or closed curvilinear, ovoid or circular shape; the groove is of the closed type. Furthermore, in embodiments, the receptacle has a central pillar surrounded by said groove (this is a so-called annular groove). In a particular embodiment, the central pillar has a constriction at its lower part.

Claims

Demands

1. A cell culture device comprising a cell receptacle (2) resting on a base (10), characterized in that the receptacle (2) is made in one piece and has a first face (30) resting on the base (10) and a second face (40) substantially opposite the first face (30), as well as a cavity (9) having an opening (8) in the second face (40) and a bottom (50) near the first face (30), which cavity (9) includes at least one groove (4) disposed in the bottom (50) and forming a culture portion arranged to receive cells (7) and allow their culture, and a guide (60) comprising at least one inclined plane (3) connecting the second face (40) to the groove (4), which inclined plane (3) has a surface texture arranged to guide cells deposited on the second face (40) substantially at the groove (4) into said culture portion by gravity like a funnel.

2. Device according to claim 1, wherein the inclined plane 3 surrounds the groove 4.

3. Device according to any one of the preceding claims, wherein the groove (4) is arranged along a closed path in the cavity (9).

4. Device according to any one of the preceding claims, wherein the groove (4) is arranged in an annular shape in the cavity (9).

5. Device according to any one of claims 3 and 4, further comprising a central pillar (6) disposed in the center of the groove (4).

6. Device according to claim 5, wherein the central pillar (6) is surrounded by the groove (4) and wherein at least a part of the periphery of said central pillar (6) defines the internal perimeter of the groove (4).

7. Device according to any one of claims 5 and 6, wherein the central pillar (6) is arranged in a general S-shape.

8. Device according to any one of the preceding claims, made at least partially, preferably totally, of a transparent material.

9. Device according to claim 8, wherein the material has a refractive index between 1.32 and 1.45, preferably equal to 1.33, so as to allow the acquisition of in situ images of individual cells or of an aggregate of cells.

10. A device according to any one of the preceding claims, made at least partially, preferably entirely, of a flexible or soft biocompatible hydrogel-based material for measuring force contraction of the aggregate of cells forming the ring-shaped tissue.

11. Device according to any one of the preceding claims, having a surface roughness Ra between 0.2 pm and 0.3 pm.

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