Cell culture device

Abstract

The invention relates to a cell culture device comprising a receptacle (2) for cells which rests on a base (10). The receptacle (2) is made in one piece and has a first face (30) which rests 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) at the level of 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) which includes at least one inclined plane (3) which connects the second face (40) to the groove (4), which inclined plane (3) has a surface condition arranged to guide cells deposited on the second face (40) substantially at the level of the groove (4) into said culture portion by gravity in the manner of a funnel.Abbreviation figure: Figure 1.

Description

Title of the invention: Cell culture device

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

[0002] In vitro procedures using cells are increasingly common in research, particularly in the initial stages of developing new molecules or drugs (preclinical testing). Their use is essential to avoid the widespread use of animal testing (in vivo testing), especially since such techniques offer numerous advantages such as ease of use, low cost, and rapid implementation.

[0003] The critical aspect of these in vitro procedures is the cells and their response to the tested molecules, drugs, or external stimuli. Although very useful, most procedures used today rely on the use of two-dimensional cell monolayers. In these situations, cells are cultured on flat, rigid substrates, for example, multi-well cell culture plates (of the ELIS A type). When comparing the 2-D in vitro environment of cells with the in vivo environment, the conclusion is reached 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 of clinical trials of new drugs.

[0004] This is why there is currently a need for in vitro models that accurately replicate 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 tested cells to respond in a way that more closely resembles reality.

[0005] Spheroids are a promising three-dimensional cell culture model for reflecting the in vivo environment of cells. These multicellular aggregates are self-assemblies of unorganized cells. For some years now, tumor cell spheroids have been considered capable of approximating the complexity of tumors in vitro and of being used for the characterization of new treatments. However, these structures have been found to have a number of disadvantages. First, spheroids are difficult to manipulate and characterize without disrupting their self-assembly. Acquiring images of the cells is difficult. inside the spheroid without manipulating it. Furthermore, for many organs, the natural organization of the organ's cells within a simple spheroid does not allow for the reproduction of the organization observed in vivo. The cells at the core 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 to cells. The effect of the drugs being tested is evaluated by verifying whether a spheroid self-assembles 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-shaped wells degrade the imaging quality of the spheroids. Furthermore, 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 displacing 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. Furthermore, the adaptability of these procedures to large-scale rapid drug screening platforms is of paramount importance.

[0009] US 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 force the cells into a ring-like arrangement. The magnetic field is removed after the cells have self-assembled in 3D. The compounds to be tested are added to the wells, and the acquired images allow the contractility of these cell rings to be quantified. The images are compared before and after the addition of the compounds to be tested.

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

[0011] WO 2017-008699, entitled "Device for measuring cell tensile force, measurement method and preparation method," describes a measurement device using a layer of nanopillars of known Young's modulus on top of which cells are cultured. Image analysis reveals the deformation of the nanopillars, and thus a map of the forces applied by each cell can be established. This method is compatible with high-resolution imaging, allowing for an understanding This reveals 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 being studied. Furthermore, the fabrication of nanopillars is expensive and complex. This prevents the adaptation of these methods to the 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 surrounded by a polymer that does not adhere to cells. Cells are cultured on these substrates and are located exclusively in the adhesive areas. The traction is quantified by comparing the images of the micropatterns with and without cells on the patterns. The analysis can then be performed by comparing the shape of the fluorescent patterns.

[0013] This micro-pattern-based method is geared towards measuring individual cells. This does not allow for the reproduction of the natural environment of cells, which is 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 only weakly representative of the 3D organization of cells in vivo.

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

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

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

[0017] The way cells organize themselves, their 3D geometry, and the geometry of their microenvironment are important factors from a biological point of view because they can impact cell maturation, physiology, and functional behavior. For example, the rate and nature of differentiation Induced pluripotent stem cells can be dependent on these factors and this has an impact on cell behavior and therefore on the representativeness of experimental results. Furthermore, the representative measurement of certain physical quantities, such as cell contraction force, requires that these cells be organized similarly to that found in a living organism, for example, into fibers for muscle cells.

[0018] The culture of 3D cells in currently known systems, in particular the culture of cells having contractile activity, is limited for the following reasons: - Cells can take a long time to organize themselves into 3D structures, and their organization is generally not representative of that observed in vivo. - A large number of cells are often lost during the process - The cells may need to be modified before being cultured (for example, magnetizing the cells with nanobeads) - The organoid produced is not always maintained, which makes it difficult to replace the surrounding environment. - The compatibility of processes with drug screening platforms is not always possible, particularly 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 the manipulation of 3D structures. - The results are limited or difficult to use (qualitative or single-cell observations) - The measurement of parameters such as the contractile force of cells is not always measurable.

[0019] The invention improves the situation. In particular, the invention avoids the drawbacks of currently known 3D systems by proposing 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.

[0020] Thus, the invention relates to 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 near the first face, which cavity includes at least one groove disposed in the bottom and forming a culture portion arranged to receive cells and allow their culture, and a guide which includes at least one inclined plane which connects the second face to the groove, which inclined plane has a surface condition arranged to guide cells deposited on the second face substantially at the level of the groove into said culture portion by gravity in the manner of a funnel.

[0021] In particular embodiments: - the inclined plane can surround the groove; - the groove can be arranged along a closed path in the cavity; - the groove can be arranged in an annular shape within the cavity; - the device may further include a central pillar located in the center of the groove; - the central pillar can be surrounded by the groove and at least part of the perimeter of said central pillar can 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 totally, from a transparent material; - the material of the device may have 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 an aggregate of cells; - the device can be made at least partially, preferably entirely, from a flexible or soft biocompatible hydrogel-based material that allows for the measurement of the contraction force of the cell aggregate forming the ring-shaped tissue; and - the surface roughness Ra of the device can be between 0.2 and 0.3 pm.

[0022] In general terms, the cell culture device described here is three-dimensional. It has a structured substrate comprising a hollow cavity shaped like a crater, the bottom of which has a groove in the form of a path in which cells can spontaneously organize themselves into a continuous three-dimensional tissue shaped along the path. The path can be of any shape, provided that the groove it defines has an aspect ratio (length of the path divided by the width of the groove) greater than or equal to 2, which will lead to the formation of a cell aggregate in the shape of the groove defined by the path.

[0023] 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.

[0024] Preferably, the pathway can be closed (ring, polygon, or any other closed irregular pathway) if the cells are contractile. To facilitate analysis, the pathway may advantageously take the form of a ring forming an annular groove in which the cells can spontaneously organize themselves into a continuous, three-dimensional, annular tissue.

[0025] The groove is flanked on at least one side and along its entire length by inclined sides whose slope points towards the groove and whose width is at least equal to the width of the groove. Preferably, these sides have an angle greater than 30° with respect to the horizontal. Preferably, the sides are of the same width all around the perimeter of the path.

[0026] When the cells are seeded on the top of this substrate, the cells fall to the bottom of the substrate under the effect of gravity or an artificially applied force such as a centrifugal force so that they are guided along the sides towards the groove, leading to the formation of a cell aggregate or tissue whose shape corresponds to that of the groove.

[0027] The structured culture substrate is preferably composed of a flat support on which grooves have been formed (preferably molded) in which the cells spontaneously organize themselves into a tissue.

[0028] According to a preferred mode, the opening of the cavity is circular and has a diameter greater than the diameter of the annular groove.

[0029] Preferably, the walls of the cavity completely surround the annular groove.

[0030] According to a preferred embodiment, the device has a central pillar disposed in the center of the groove when the latter is annular in shape.

[0031] According to one embodiment, the central pillar is completely surrounded by the annular groove and preferably defines the inner perimeter of the annular groove. Advantageously, the central pillar has a local reduction in its perimeter at an intermediate height to ensure reliable and constant maintenance of the cell ring's position around the pillar, particularly 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 in perimeter prevents the ring from sliding along the pillar if it contracts around it.

[0032] According to a preferred method, the local perimeter reduction is less than 20% of the average perimeter of the central pillar to facilitate demolding of the structure.

[0033] Thus, the central pillar can take on a general S-shape. This S-shape allows for reliable and constant maintenance of the cell ring's position around the pillar, particularly when the cells contract. As explained above, this local reduction in perimeter prevents the ring from sliding along the pillar; advantageously, maintaining the ring allows for reliable and reproducible observations and measurements over time.

[0034] According to a preferred embodiment, the local perimeter reduction is less than 20% of the average perimeter of the central pillar to facilitate 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 conical in shape to guide the seeded cells towards the groove.

[0035] According to another preferred embodiment, the device is made at least partially, preferably totally, of transparent material, preferably with an optical 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.

[0036] According to a preferred embodiment, the device is made at least partially, preferably entirely, of a flexible or soft material that allows the contraction force of the cell aggregate forming the annular tissue to be measured. The behavior of the central pillar can be likened to that of an elastic solid. Thus, the contraction force, (F), exerted by the 3D cellular structure is proportional to the variation of the pillar radius (Ar) multiplied by a constant that depends on the mechanical properties of the material, such as its elastic modulus or Young's modulus (-F ≤ Ar).

[0037] 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 Newtons), R is the radius of the pillar at the point where the ring encloses it (in meters) and h is the height of contact between the ring and the pillar (in meters)

[0038] Preferably the soft or flexible material is a bio-compatible material or a hydrogel allowing the diffusion of nutrients and / or oxygen and / or allowing the measurement of the contraction force of the ring-shaped cell aggregate.

[0039] According to one embodiment, the device is made partially or totally of a photopolymerizable polymer material in order to reduce its manufacturing time.

[0040] In order to allow the cells to concentrate in the ring-shaped groove, the walls of the cavity are made of a non-adherent material for the cells in order to allow the cells to freely exert their own force on the aggregate.

[0041] 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 helps to stabilize its shape and prevent the cell aggregate from contracting on itself and taking the form of a simple spheroid.

[0042] 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.

[0043] 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.

[0044] According to another aspect, the invention also relates to a method for manufacturing a device as described above, characterized in that it includes, in particular, the following steps: - Prepare a substrate preferably made of a transparent material (e.g. glass or plastic, for example in the form of a petri dish, multi-well plate or glass coverslip) - Deposit a layer of an unpolymerized monomer solution onto 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 in order to create a structured substrate - Polymerize the monomer solution of said layer in such a way as to harden the structured substrate - Remove the hardened structured substrate from the mold.

[0045] Finally, the culture device according to the invention, the multi-well support and the method can be used for the production of cell culture, in particular of cells derived from human cells.

[0046] 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.

[0047] 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) by the presence of the (preferably inclined) flanks of the cavity, which ensure the guidance and sliding of the cells within the groove. Such a device therefore has a structure that generates a robust and reproducible technique for assembling cells into 3D structures.

[0048] 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 the organoid. This makes it possible, 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 exhibiting contractile behavior, and also to measure changes due to the effect of specific compounds, the chemical environment of the cells, or cell modification.

[0049] 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 the surface tension of the cell aggregate, particularly for non-contractile cells. For example, to indirectly measure the surface tension of an aggregate of epithelial cells.

[0050] To measure the contraction force of the cells in the organoid, the measurement of the variation in diameter is used to evaluate the variation in the contraction force applied by the cell structure on the central pillar.

[0051] For small deformations, dimensional analysis shows us that the force applied by the organoid can be determined as follows:

[0052] dF = dO.E.dD

[0053] where dF is the variation of force in Newtons, dD the variation of 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.

[0054] Advantageously, an active ingredient or drug such as Latrunculin A can be used to inhibit cell contraction and thus deduce the strength of contraction before application of the drug.

[0055] The device can also be used to measure contraction force profiles over time by recording films 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.

[0056] 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 organoids compared to spheroids.

[0057] Yet another use of the device according to the invention consists of creating 3D cell aggregates in a single seeding step. The cells remain trapped on the device due to the contraction of the ring-shaped organoid around the pillar it surrounds. This allows the culture medium around the organoid to be replaced without disturbing it.

[0058] 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.

[0059] Monomer molecules react in such a way as to form a three-dimensional network or polymer chain by polymerization and forming polymers which can be of synthetic or natural nature.

[0060] Preferably, the polymer will be a hydrogel allowing the diffusion of nutritional molecules and the oxygenation of the organoid and therefore its well-being.

[0061] Below are a number of monomers used to generate polymeric materials (gels and in particular hydrogels)

[0062] -Poly (2-Hydroxyethyl methacrylate)- PHEMA

[0063] -2-Hydroxyethyl methacrylate - HEMA

[0064] -Polyethylene glycol (PEG)

[0065] -Methacrylic acid (MAA)

[0066] -Acrylamide / acrylic acid copolymer

[0067] -Poly(N-isopropylacrylamide)

[0068] -Peptide-based polymers that are not hydrogels can also be used, or elastomers such as silicones (for example polydimethyl siloxane) with which it is easy to mold such shapes.

[0069] Preferably this polymer will be photo-polymerizable to accelerate the manufacturing process.

[0070] Ideally, the polymer will be optically transparent (refractive index close to that of water, 1.33) after polymerization, which allows for optical microscopic imaging (e.g., fluorescence, high-resolution imaging, etc.). This allows for results based on image analysis.

[0071] The material's rigidity can be adjusted to suit the cell environment. Therefore, materials with a rigidity between 0.1 kPa and 1 MPa, preferably between 1 and 20 kPa, will be chosen.

[0072] 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 the organoid from adhering to the substrate. In cases where adhesion is desired, a material that allows cell adhesion will be used. This can be further improved by the presence on the surface of a specific extracellular matrix such as collagen, fibronectin, etc.

[0073] The dimensions of the annular groove can be adapted to the size of the organoids used in the process within the limits described above.

[0074] The invention will be better understood with the aid of the following embodiments in conjunction with the figures which represent:

[0075] [Fig-1] shows a cross-sectional view of a device according to the invention;

[0076] [Fig.2] shows a top view of a device according to the invention;

[0077] [Fig. 3] shows a cross-sectional view of a device according to the invention with respective dimensions;

[0078] [Fig.4] shows molding steps of a device according to the invention;

[0079] [Fig. 5] shows photographs of molds for manufacturing the device according to the invention;

[0080] [Fig.6] shows a photograph of fibroblast cells after centrifugation;

[0081] [Fig.7] shows a photograph of the cells of [Fig.6] one hour after centrifugation and polymerization of collagen;

[0082] [Fig.8] shows a cross-sectional view of an alternative embodiment of the device according to the invention;

[0083] [Fig.9] shows a cross-sectional view of a variant of the device according to the invention with their respective dimensions;

[0084] [Fig. 10] shows a photograph of MEF cells 22 hours after culture in the ring and 12 days after; and

[0085] [Fig. 11] shows a diagram of the contraction and relaxation of a central pillar caused by tension cells arranged around the pillar.

[0086] The figures, tables, and description below essentially contain elements of a certain nature. The figures and tables form an integral part of the description and may therefore not only serve to better understand the present invention but also contribute to its definition, if necessary.

[0087] The invention is now described with reference to figures 1 to 11. In the figures, the same elements bear the same references.

[0088] Figure 1 shows a vertical cross-sectional view of the structured cell culture substrate 1 according to the invention. The substrate comprises a receptacle 2 resting on a base 10. The substrate further comprises a groove 4 having an annular shape. To give this annular shape to the groove 4, a central pillar 6 is arranged, surrounded by the groove 4. This central pillar 6 preferably has a conical apex 5. 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 wall structure 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 cavity 9 connects the inner edge of the opening 8 located at the top of cavity 9 to the outer edge of the annular groove 4 located at the base of cavity 9. More specifically, cavity 9 includes groove 4, which is disposed in a bottom 50 of the cavity. Groove 4 forms a culture portion arranged to receive cells 7 and allow their culture. For this purpose, cavity 9 further includes a guide 60 which comprises at least one inclined plane 3. Inclined plane 3 connects the second face 40 to groove 4. According to the invention, inclined plane 3 has a surface texture arranged to guide cells deposited on the second face 40 substantially to the level of groove 4. The cells thus migrate into the culture portion by gravity in the manner of a funnel.

[0089] When the cells are placed on the structured substrate 1, or more precisely on the second face at the level of a guide 60, the funnel-shaped wall structure guides the cells towards the annular groove 4, leading to the spontaneous formation of a three-dimensional aggregate of annular cells. For this purpose, the guide 60 includes an inclined plane 3. Optionally, the conical tip of the central pillar 5 helps the cells 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 apex angle of the funnel.

[0090] In [Fig.1], we can see the cells 7 represented schematically which partially overlap to form the annular aggregate.

[0091] The dimensions of the annular groove can vary widely to facilitate the formation of cell aggregates according to the invention. Referring to [Fig. 3], the depth hl of the annular groove can 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 W1 between 3 and 3000 microns, preferably between 50 and 200 microns.

[0092] The cavity 9, preferably having a conical funnel shape along its walls, shall 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, thus reducing the number of cells in the device. The apex angle Alpha 1 may 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.

[0093] The central pillar, preferably defining a vertical axis of symmetry of the device according to the invention, shall have a height h3 between 1 and 4000 microns, preferably between 150 and 550 microns, a diameter d2 between 1 and 5000 microns, preferably between 100 and 350 microns, a vertical part of height h4 between 1 and 3000 microns, preferably between 60 and 150 microns, and an angle at the top Alpha 2 between 0 and 180 degrees, preferably between 80 and 100 degrees, (an angle of zero or 180 corresponding to a pillar with a flat top), of height hl.

[0094] 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 that terminates 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. 1], 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 that can slide upwards under the action of the cells, while remaining fixed to the central pillar thanks to its geometric shape forming a protuberance 16 in [Fig. 8].

[0095] 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.

[0096] Cells can be cultured in two ways: either simply suspended in a cell culture medium, or mixed with a natural or synthetic extracellular matrix, such as, for example, type I, II, III, IV, and V collagen. Collagen is the main component of the natural extracellular matrix in the human body; this protein is present between cells in many connective tissues. It can also be a matrigel, a hydrogel, or other extracellular matrices.

[0097] The concentration or quantity of cells that must be seeded on the substrate depends on the type of cells and the diameter of the cavity compared to the surface of the ring.

[0098] The fabrication of the structured substrate can be carried out simply by molding using a mold allowing the desired structure to be imprinted (see for example below [Fig.4]) in the material used.

[0099] Since this molding process is simple, it is easy to mold a plurality of devices in a single step to produce multi-well plates.

[0100] Figure 4 illustrates a method for manufacturing the device according to the invention. A flat support 23 (preferably a Petri dish, glass plate, multi-well plate, etc.) is coated with a layer 24 of the chosen polymer (in its unpolymerized state) in liquid or soft form, onto which the microstructured substrate 1 is printed using a mold 20 having the negative shape of the structured substrate 1. The polymerization reaction then starts, and the coated layer 24 takes the desired shape of the positive structure of the microstructured substrate 1. After demolding (Fig. 4), the final 3D microstructured substrate 1 is obtained.

[0101] Ideally, the mold surface will preferably be very smooth with a roughness of less than one micron. Therefore, the molds will preferably be manufactured using high-resolution processes (CNC milling, 3D printing using two-photon lithography).

[0102] The device according to the invention allows the use of any type of biological cells that can adhere to one another, such as endothelial cells, cardiac cells, smooth muscle cells, epithelial cells, chondrocytes, fibroblasts...

[0103] 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.

[0104] Monomer molecules react in such a way as to form a three-dimensional network or polymer chain by polymerization and forming polymers which can be of synthetic or natural nature.

[0105] Cells can be cultured in two ways: either simply suspended in a cell culture medium, or mixed with a natural or synthetic extracellular matrix, such as, for example, type I, II, III, IV, and V collagen. Collagen is the main component of the natural extracellular matrix in the human body; this protein is present between cells in many connective tissues. It can also be a matrigel, a hydrogel, or other extracellular matrices.

[0106] The concentration or quantity of cells to be seeded on the substrate depends on the type of cells and the diameter of the cavity compared to the surface of the ring.

[0107] Figure 5 represents a mold for producing a plurality of devices according to the invention.

[0108] The manufacture of a mold of the invention is however accompanied by numerous technical problems which the Applicant had to overcome.

[0109] Microfabrication or micromachining plays a major role in the manufacture of the device of the invention. In technical fields unrelated to the invention, such as the semiconductor industry or microfluidic research, miniaturization has seen technological advances in recent years. However, the transposition of this miniaturization to the field of the invention is generally impractical or even incompatible.

[0110] The Applicant thus initially turned to photolithography, a technique used to transfer a certain shape or pattern onto a material or surface by selectively exposing light-sensitive polymers using a photographic mask. "Dry etching" or "wet etching" methods can then be used to remove the material from the previously selected areas.

[0111] Wet etching is a chemical process that is relatively simple and quick to implement. The material can be etched isotropically (orientation-independent) or anisotropically (orientation-dependent). Isotropic etching is the most common. In this method, the material is removed uniformly in all directions, which can result in material being removed from areas that should not be etched. Anisotropic etching (when using crystalline substrates) removes less material from under the mask, allowing for better control of the wall geometry. Dry etching techniques use ion bombardment at 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.These two techniques are commonly used, but are heavily limited to simple designs, such as straight or vertical walls.

[0112] In addition, 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 specific techniques, such as material extrusion, laser sintering, material jetting, etc. All these techniques are attractive because they do not require special tools or Modifications to the process allow for relatively low initial costs. Furthermore, these techniques can be used to fabricate rather complex shapes and features, but it's important to remember that 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 (nanometer scale) based on two-photon polymerization, challenge the limitations of conventional additive manufacturing methods. However, they are only suitable for fabricating very small structures. These structures are smaller than the dimensions required to create a mold that maximizes the surface area used in standard cell culture substrates, such as typical 96-well plates (a 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. However, this technique is primarily used in research, with very high costs and long manufacturing times. Additive manufacturing methods are therefore not suitable for manufacturing the molds that will produce the cell culture devices described in the present invention. It is difficult, using additive manufacturing methods, to produce structures with a good surface finish, and therefore very high resolution, in a reasonable timeframe, and with a large volume, which is necessary to create the device according to the invention allowing for the simultaneous production of a large number of organoids.

[0113] Computer numerical control (CNC) machining is a subtractive technique. It starts with a block of material, for example plastic or metal, which is shaped using specific tools such as drills. It offers high dimensional accuracy and allows the production 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 (degrees of freedom – in particular, 5 axes), as well as the speed and sensitivity of the response or operation. 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 needs to be precisely adjusted.

[0114] All these techniques come from technical fields far removed from the present invention. EXAMPLE OF AN INVENTION IN IMPLEMENTATION

[0115] 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.

[0116] To select a suitable method for manufacturing the molds of the invention, the Applicant considered numerous parameters and constraints, including in particular:

[0117] - The use of a robust material that can be used multiple times for molding The polymers chosen include polyacrylamide or polyethylene glycol hydrogels. The material must also withstand harsh cleaning procedures, which may be necessary to remove residues from molded materials;

[0118] - A material that does not react and / or that adheres to the polymers in question;

[0119] - A method that allows manufacturing at the micron scale and is not limited with straight characteristics, but which can adapt to complex shapes such as "s" or curved profiles, inclined walls in particular;

[0120] - The need for surfaces with a very smooth finish, necessary both to facilitate The demolding process is designed to avoid reproducing a rough surface in the final cell culture device. This is necessary to ensure that the cells are not trapped by micro-roughness on the polymer surface, but rather guided by the angled walls towards the cell assembly area.

[0121] In view of the above, a process based on material subtraction proved, somewhat surprisingly, to be the most suitable for manufacturing a mold such as that shown in [Fig. 5]. The manufacturing process included, in particular, the following steps:

[0122] - The selection of a stainless steel material (grade 1.4305), in order to meet at least some of the aforementioned requirements;

[0123] - A two-phase machining process, a first phase in which a laser machine 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 create the cavity that will give rise to the central pillar;

[0124] - A laser machining process for material removal, followed by micro-scanning drill. Indeed, laser machining does not allow for a satisfactory surface finish (the roughness exceeded the micrometer scale). Only after scanning with a micro-drill over the entire or nearly entire surface are thin layers of excess material removed until the surface is sufficiently smooth, i.e., with a roughness Ra between 0.2 µm and 0.3 µm. The technique employed allows for edges with a curvature of approximately 0.1 mm.

[0125] The mold of the invention is thus made of stainless steel having a surface roughness Ra between 0.2 and 0.3 pm. It thus makes it possible to obtain a device of the invention having a roughness Ra between 0.2 and 0.3 pm.

[0126] - The internal cavity is made in two stages: a first stage during which A drill bit with a diameter smaller than that of the central pillar is used to drill a cylindrical hole, and a second step during which a spherical profile drill bit is used to fine-tune the profile of the walls, making it possible to create "S" shaped or curved profiles and a sharp conical hole that gives the conical profile of the central pillar.

[0127] High-resolution computer numerical control (CNC) machining is a flexible technique. To implement the above manufacturing process, numerous details are taken into account and must be kept in mind when manufacturing a cell culture device of the invention.

[0128] The following example uses a polyacrylamide-based substrate.

[0129] -Mold manufacturing:

[0130] The manufacture of the mold for producing the device according to the invention is shown in Figure 5a (cross-sectional view) and Figure 5b (top view). An internally shaped mold, which is the negative of the structured substrate, is used to mold a structure as shown in [Fig. 1]. The mold is made of stainless steel (grade 1.4305 in this example) produced by high-resolution CNC milling with an average surface roughness Ra 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], the negative shape of the molds relative to the shape of the device is noted, with the annular groove (in negative) located above the conical structure of the walls: at the center of the annular groove mold, there is a deeper cavity corresponding to the central pillar after molding.

[0131] An acrylamide-based hydrogel was then used to mold the devices in a sterile environment.

[0132] -Preparation of the support:

[0133] The starting support consists of 16 mm diameter glass strips which are cleaned and silanized to promote adhesion.

[0134] -Pre-hydrogel solution:

[0135] After coating the pre-hydrogel solution (acrylamide solution), two different mechanisms can be used to trigger (as catalytic agents) the polymerization of this hydrogel:

[0136] - by using UV radiation of an appropriate wavelength (e.g., 365 nm) which catalyzes the polymerization of acrylamide and bis-acrylamide free radicals 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);

[0137] -using ammonium persulfate (APS) to induce polymerization (by decomposing it forms free radicals) of acrylamide and bisacrylamide monomers and using a free radical stabilizer (TEMED) which is a polymerization promoter.

[0138] 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.

[0139] In this particular case, 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).

[0140] -Moulding (polyacrylamide):

[0141] The treated glass slides are coated with a drop of 15 microlitres of pre-hydrogel solution.

[0142] The mold is placed above the lamellae, 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 lamellae thus prepared are assembled on a multi-well plate.

[0143] The mold used comprised 23 microstructures allowing the fabrication of 23 cavities with annular grooves, thus enabling several tests to be carried out in the same well. The width (wl) of the rings is 100 pm and the diameter of the central pillars is 240 pm.

[0144] Cell culture using fibroblasts derived from human abdominal skin cells:

[0145] The study of fibroblast contractility is currently an important topic for understanding the phenomena of inflammation and healing.

[0146] -Fibroblast cell protocol:

[0147] Fibroblasts derived from human abdominal skin cells were resuspended in an extracellular matrix of purified type 1 collagen with a collagen concentration of 160,000 cells / ml. A 15 microliter drop of the cell mixture was deposited onto the top surface of the structured substrate, which had been previously incubated in cell culture medium. The substrate was then centrifuged for 2 minutes at 100 G. The cells are naturally guided to the annular groove by the centrifugal acceleration. The collagen mixture was then polymerized at 37°C for 1 hour, followed by the culture medium. cellular material is added to the wells. [Fig.7] is an image of 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 form a perfect ring around the central pillar.

[0148] This example shows the rapid and spontaneous assembly of a 3D organoid in annular shape using the device of the invention.

[0149] - MEFs (Mouse Embryonic Fibroblasts) cell protocol:

[0150] Structured hydrogel-based substrates with an "S"-shaped central pillar were fabricated using stainless steel molds. The ring width (wl) was 70 µm and the central pillar diameter was 360 µm. The hydrogel used was a 6.4% polyethylene glycol-based hydrogel, UV-cured.

[0151] MEF cells are first dissociated from their substrate using trypsin and then centrifuged. The cell pellet is then resuspended in fresh cell culture medium at a concentration of 160,000 cells per ml. Fifteen 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 maintained in culture for 12 days, and the medium is changed every two days. After 12 days, the aggregate of MEF cells has formed a more compact ring-shaped structure, as shown in [Fig. 10].

[0152] This example demonstrates the rapid self-assembly of ring-shaped organoids. Since no extracellular matrix was used in this case, the cells self-sediment onto the ring-shaped cavity, and no centrifugation step was required. In this case, the funnel-shaped structure guides the cells into the ring-shaped cavity.

[0153] This example also demonstrates that the organoid can be preserved in structured substrates for a long period. 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 potentially slide along the central pillar, or when the liquid is agitated, for example, when changing the medium. The organoid's secure positioning allows for stable observation over a long period while also permitting easy changes to the surrounding medium.

[0154] In another example, we culture C2C12 skeletal muscle cells in the form of ring-shaped organoids and measure their contraction strength.

[0155] To measure the contraction force of the cells in the organoid, we measure the deformation of the central elastic pillar under the effect of the force.

[0156] The measurement of the variation in the diameter of the pillar (in other words, the inner diameter of the ring) is used to evaluate the variation in the contraction force applied by the cellular structure on the central pillar.

[0157] Advantageously, a drug such as Latrunculin A (an inducer of actin depolymerization) can be used to inhibit cell contraction and thus deduce the strength of contraction before application of the drug.

[0158] The gels used in this example follow the same manufacturing protocol as the previous example with MEFs.

[0159] C2C12 cells are dissociated with trypsin and then centrifuged. The cell pellet is then resuspended in fresh culture medium at a concentration of 500,000 cells per ml. 200 µl of this cell suspension are 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.

[0160] After 6 hours, the structures are imaged and the diameter of the central pillars is measured. To assess the force applied by the organoid on the central pillar, the cells are exposed to a medium containing Latrunculin A (1:1000). After 30 minutes of incubation, the structures are imaged again and the diameter of the central pillars is measured.

[0161] In our example, the diameter of the central pillar increases by approximately 1.5% in the presence of Latrunculin A compared to the control experiment without the drug. This is demonstrated in [Fig. 11].

[0162] In particular embodiments: the groove has a bottom and two sides; the groove is arranged in a straight, open or closed curvilinear, ovoid, or circular shape; the groove is of the closed type. Furthermore, in some embodiments, the receptacle has a central pillar surrounded by said groove (this is a so-called annular groove). In one 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. Device according to any one of the preceding claims, made at least partially, preferably totally, of a flexible or soft biocompatible hydrogel-based material enabling the measurement of the contraction force of the aggregate of cells forming the annular-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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