Device for cell or tissue culture

Abstract

The invention relates in particular to a cell culture device comprising an insert (1) having an internal cavity (11) delimited by an insert side wall (12) and an insert bottom (10), said internal cavity being able to receive said cell culture in a cell culture fluid so as to create a cell tissue (2). The device comprises a container (3) that removably receives said insert, said side wall (12) having first through-holes (13). The container comprises an internal flue (30) and an opening (33) which allows said insert (1) to be inserted into said flue. The insert bottom comprises second through-holes (14) allowing observation of the cells therethrough, said first and second through-holes being capillary holes able to retain said cell culture fluid in said internal cavity and serving to anchor said cell tissue.

Description

[0001] DESCRIPTION

[0002] TITLE: DEVICE FOR CELL OR TISSUE CULTURE

[0003] FIELD OF THE INVENTION

[0004] The invention relates to a device for cell or tissue culture, comprising a container and an insert. The insert is capable of being placed in said container and capable of receiving cells or tissues to be cultured.

[0005] Cell culture and tissue culture are considered to be two types of processes used for growing cells of multicellular organisms outside their natural environment.

[0006] The goal of cell culture is to grow cells outside the body. Its uses are important: it allows for pharmacological and chromosomal studies, it helps in basic research, antibody production, protein synthesis, etc.

[0007] STATE OF THE ART

[0008] There is a strong need to carry out experiments on cell or tissue culture samples in various fields of activity, notably cosmetics and / or pharmaceuticals.

[0009] For example, there is a strong need to study and develop therapies for healthy and diseased skin. These therapies increasingly use skin-equivalent models on which products are tested.

[0010] These skin-like models are increasingly in demand and are being created following the European Union's complete ban on animal testing.

[0011] Skin equivalent models are created by growing cells in devices, and come in the form of artificial skin tissue, for example.

[0012] The methods used allow the creation of artificial skin tissues. The goal is to create artificial skin tissues, or artificial cells, that have properties similar to those of native human skin, both structurally and functionally. More generally, the goal is to create soft tissues, for example skin tissues but also liver, kidney, lung, adipose, pancreas, etc.

[0013] A known way to create these artificial skin tissues is to use cell culture inserts. These inserts are designed to allow static cell culture in which skin cells are matured without stressful environmental conditions until the tissue of the desired size and thickness is obtained.

[0014] Document WO2011127945 describes, for example, a cell culture system using inserts that are placed in a container and into which cells or tissues are introduced to be cultured. Each insert makes it possible to create a cell tissue that is subsequently used for experimenting with cosmetic or pharmaceutical products, for example. In the example presented in document WO 2011127945, the inserts each have a side wall and a bottom wall, the side wall having through openings through which a liquid can pass. These inserts are placed in wells, arranged in a support. A liquid, allowing the cells to be cultured, is introduced into the wells.

[0015] Cell tissues are obtained after a certain time, this time being that which is necessary for the tissues or cells to develop in stress-free environmental conditions.

[0016] The invention was originally designed to improve the properties of the resulting artificial skin, i.e. to improve the properties of the cell tissue so that it is even closer to natural skin tissue: in fact, until now, the artificial skin tissues created do not really imitate certain natural mechanical resistance properties that natural skin exhibits during its development.

[0017] It is known that natural growth of human skin occurs simultaneously when the human body is exposed to multiple environmental stresses and conditions: It has been observed from several studies that when fibroblast cells are exposed to different types of stress, it results in an increase in collagen production due to the activation of several signaling pathways in the cell in response to the stress exposed on the surrounding collagen protein.

[0018] The production of collagen proteins provides a certain mechanical strength to the overall structure of the skin. It is these natural mechanical strength properties that the invention aims to achieve in the artificial skin tissues created.

[0019] Some studies have successfully developed skin maturation bioreactors by inducing stress using multiple techniques. However, these developed bioreactors are very complex to construct and are not commercially available. There is no dedicated bioreactor available on the market yet for dynamic maturation of skin tissues.

[0020] PRESENTATION OF THE INVENTION

[0021] The invention therefore aims to provide a device that makes it possible to obtain cell tissues, for example a tissue imitating natural skin by having mechanical properties similar to those of natural skin. The invention also aims to provide a device that makes it possible to better visually (or by microscopy) control cell development while avoiding manipulations of the culture itself (in a non-destructive manner).

[0022] The invention relates to this purpose to a cell culture device comprising an insert having an internal cavity delimited by an insert side wall and an insert bottom, said cavity being capable of receiving said cell culture in a cell culture fluid (for example a hydrogel) so as to create a cell tissue, said device comprising a container in which said insert is received in a removable manner, said insert side wall having first through holes.

[0023] The device according to the invention is remarkable in that said container which receives the insert comprises an internal chimney, delimiting an internal wall of said container and forming a conduit having a first opening at a first end of the conduit and a second opening at a second end of the conduit, opening into a bottom of the container, said first opening end allowing the insertion of said insert into said chimney of the container, said bottom of the insert comprising second through holes allowing the observation of the cells in culture through the bottom of the insert by the second opening of the second end of the conduit of said chimney, said first and second through holes being capillary holes capable of retaining the cell culture fluid in said internal cavity of said insert and serving as anchoring to said cell tissue.

[0024] Indeed, and according to a first technical function that the invention allows, the through openings which are in the form of capillary holes serve to anchor the cell culture fluid in the insert, which makes it possible to obtain a tissue of cells which adheres to the wall of the insert, to ensure tension on the tissue to avoid its retraction (the phenomenon of retraction is a phenomenon classically observed on tissues in culture, over the days of culture: the invention avoids this phenomenon). In addition, according to a second technical function that the invention allows, the opening in the bottom of the chimney, in which the insert is placed, allows the bottom of the insert to be seen and the capillary through holes allowing the cell culture to be observed through the bottom of the insert without however allowing the cell culture to "leak" through the bottom of the insert.

[0025] By "capillary" we mean that the holes are small enough to prevent the cell culture fluid, received in the cavity of the insert, from passing through them thanks to the surface tension of the cell culture fluid.

[0026] The invention thus allows monitoring of the evolution of the cell culture during the creation of the cell tissue and the insert allows the cell culture fluid (and the cell culture which develops therein) to anchor itself in the first and second through holes that the insert has, to allow the created cell tissue to cling to the walls of the insert (side and bottom of the insert) in order to create a tissue which is distributed in a substantially homogeneous manner in the insert with greater tensions at the anchoring points: this makes it possible to simulate an anchoring of skin on the limits of a wound, for example (by simulating the organization of a natural extracellular matrix), to maintain the initial surface of the created cell tissue, as well as its thickness and avoids the retraction of the tissue observed with the solutions of the State of the Art, as mentioned above.

[0027] The device according to the invention may also comprise the following characteristics, taken separately or in combination: Advantageously, the chimney comprises third through holes, arranged in the internal wall to cooperate with said first through holes when the insert is received in said chimney, said third through holes ensuring the passage of a feed fluid between the cavity of the insert and a tank for receiving the feed fluid, said tank being delimited between a side wall of the container, the bottom of the container and the internal wall formed by the chimney.This allows movement of the feed fluid, into which is immersed the cell culture fluid (for example hydrogel in an example to be described and illustrated later) that can be accommodated in the device during cell culture, when the device is subjected to movement, to recreate stress conditions allowing the cells to react and acquire mechanical properties close to those of real skin.

[0028] According to an embodiment described below, which is not limiting, the insert side wall comprises at least two rows of first through holes and the internal chimney comprises at least two rows of third through holes, at least one of said at least two rows of first through holes cooperating with at least one of said at least two rows of said third through holes when said insert is inserted into said chimney flue.

[0029] Advantageously, said first through holes and said second through holes have substantially the same shape and the same diameter. Preferably, the diameter of the first and second through holes is (or substantially) between 1000 and 4000 μm, preferably less than or equal to 2000 μm (or substantially 2000 μm).

[0030] According to an advantageous embodiment, it is provided that the chimney comprises first removable fixing elements of the insert, the insert being equipped with second removable fixing elements which are associated with said first removable fixing elements. The fixing of the insert in the chimney is thus carried out by means of removable fixing elements, the insert and the container being equipped with complementary means which, when associated, allow this removable fixing.

[0031] The first removable attachment elements of the insert are distributed over at least two levels in said chimney, each of said at least two levels being located between said first and second ends of said chimney. The insert can then be positioned, within the framework of this embodiment, at two different levels. It is thus possible to create different cell development layers, to reproduce the layers of the skin and to introduce an air / liquid interface during the growth of the tissue (skin) if necessary.

[0032] In a further advantageous embodiment, the insert may comprise at least one external lateral projecting tab, said projecting tab comprising at least one blind hole, and the chimney may comprise a notch arranged in an axial direction through the chimney wall, the notch having an edge having at least one projecting pin, said at least one blind hole of said at least one projecting tab constituting said at least one first fixing element of the insert and said at least one projecting pin of said chimney constituting said at least one second fixing element of the chimney, removably associating with said at least one first fixing element of the insert.

[0033] The protruding tab here serves as removable fixing means. As will be seen later, it can also serve as a gripping element for gripping the insert. Preferably, the insert comprises at least one boss on an edge formed by a free end of said insert side wall, and at least one housing provided in the vicinity of the edge of the bottom of said insert, said at least one housing having a shape complementary to that of said at least one boss, said at least one housing being capable of receiving said at least one boss of a second identical insert positioned under the insert of said device. This technical feature allows assembly of several inserts of the device on top of each other by stacking.

[0034] The invention also relates to an insert for a device as defined above. The insert according to the invention comprises an internal cavity delimited by an insert side wall and an insert bottom, said cavity being capable of receiving said cell culture so as to create a cell tissue, said insert side wall having first through holes. The insert according to the invention is remarkable in that the insert bottom comprises second through holes allowing observation of the cells in culture through the bottom of the insert, said first and second through holes being capillary holes capable of retaining said cell culture fluid in said internal cavity of said insert and capable of serving as anchors to the cell tissue.

[0035] Preferably, the first and second through holes have a diameter of (or substantially of) between 1000 and 4000 pm, preferably 2000 pm (or substantially 2000 pm).

[0036] Furthermore, the insert comprises at least one boss on an edge formed by a free end of said insert side wall, and at least one housing provided in the vicinity of the edge of the bottom of said insert, said at least one housing having a shape complementary to that of said at least one boss, said at least one housing being capable of receiving said at least one boss of a second identical insert positioned under the insert of said device.

[0037] The invention also relates to an installation comprising at least one device as defined above, as well as a support on which said at least one device can be positioned, said support being mounted movably while being associated with a movement drive apparatus, to drive in movement the feed fluid in which the culture medium is immersed in said insert during the formation of said cell tissue.

[0038] The invention finally relates to a method for producing a cell tissue, implementing an installation as defined above, said method being remarkable in that it comprises the following steps: a) positioning said insert in said chimney of said container, b) positioning said device on said support of said installation, c) introducing a cell culture fluid and cells to be cultured into the cavity of said insert, step c) being able to be carried out before step a), d) introducing a feed fluid into said device, e) actuating the apparatus for driving said support in movement at least partially for a predetermined time, to cause a movement of said feed fluid in said insert, until at least a portion of cell tissue is obtained in said insert, f) removing said device from said support, and g) extracting said insert from said container, said insert comprising said cell tissue.

[0039] Preferably, in the case where the installation comprises a microscopic observation device positioned under said support, said support being transparent, the method according to the invention may comprise an additional step d1) between step d) and step e), according to which the development of said cell tissue is observed through said second through holes of said bottom of the insert.

[0040] More preferably, in the case where the device is such that the chimney of said receiving container comprises four protruding pins from the axial edge formed in the wall of the chimney, to position said insert at four different levels in said chimney between the first and second opening of said chimney, the four protruding pins comprising:

[0041] - a first protruding pin substantially at the level of the bottom of said container, in the vicinity of said second opening, said bottom level of said container defining a first level in said chimney,

[0042] - second and third protruding pins between said second opening and said first opening of the chimney, the positioning of the second and third pins defining a second and third level of positioning of said insert in the chimney of the container,

[0043] - a fourth protruding pin substantially at the level of the first opening of said chimney, the fourth pin defining a fourth level of positioning of said insert in the chimney, the method has the following particularity: in step c), said insert is positioned at said first level of said chimney, then optionally step d) is carried out or the cells are allowed to develop, for a first predetermined time, at the end of said first predetermined time, the insert is positioned at said second level, then optionally step d) is carried out again, at the end of a second predetermined time, the insert is positioned at said third level, then optionally step d) is carried out again.

[0044] This step allows for the optional introduction of an air / liquid interface: the lower surface and the peripheral edge of the cell culture fluid are in contact with the culture medium (feeding fluid) while the upper surface of the insert is kept dry for the maturation of the keratinocytes, for example. The upper oblong-shaped hole of one embodiment, which will be described later, is intended to stop the flow of the feeding fluid due to surface tension.

[0045] Finally, and at the end of a third predetermined time, the insert is positioned at said fourth level, then step d) is possibly carried out again, or the cells are left to develop, for a fourth predetermined time. This step can allow the application of chemical compounds to be tested on the skin after its maturation.

[0046] PRESENTATION OF FIGURES

[0047] Other advantages and characteristics of the invention will appear on examining the detailed description of a non-limiting embodiment, and the appended drawings, in which:

[0048] [Fig. 1] is a perspective and top view of an insert of a device according to the invention,

[0049] [Fig. 2] is a perspective and bottom view of the insert shown in Figure 1, [Fig. 3] is a perspective view of several device inserts stacked on top of each other, [Fig. 4] is a perspective and top view of a device container according to the invention,

[0050] [Fig. 5] is a perspective and bottom view of the container shown in Figure 4,

[0051] [Fig. 6] is a perspective view showing the insert and container of a device, one above the other, the insert being adapted to be inserted into the container,

[0052] [Fig. 7] is a perspective and bottom view of the assembly according to the invention, the insert being received at a first level in the container,

[0053] [Fig. 8] is a perspective and top view of the assembly shown in Figure 7,

[0054] [Fig. 9] is a perspective and bottom view of the assembly according to the invention, the insert being received at a second level in the container,

[0055] [Fig. 10] is a perspective and top view of the assembly shown in Figure 9,

[0056] [Fig. 11] is a perspective and bottom view of the assembly according to the invention, the insert being received at a third level in the container, [Fig. 12] is a perspective and top view of the assembly shown in figure 11,

[0057] [Fig. 13] is a perspective and bottom view of the assembly according to the invention, the insert being received at a fourth level in the container, [Fig. 14] is a perspective and top view of the assembly shown in Figure 13, and

[0058] [Fig. 15] is a black and white photograph of a skin sample obtained, in the first part of the device, and

[0059] [Fig. 16] schematically illustrates an installation in accordance with the invention, as well as certain steps of a method in accordance with the invention for implementing the installation.

[0060] METHOD OF IMPLEMENTATION

[0061] Figures 1 to 3 only show one insert 1 of a device according to one embodiment of the invention.

[0062] Insert 1 serves to accommodate a cell culture and serves as a support for the cell tissue that is created there.

[0063] In accordance with the invention, the device also comprises a container 3 in which the insert 1 is removably received: the container 3 will be presented subsequently with reference to figures 4 and 5.

[0064] As shown in Figures 1 and 2, the insert 1 generally takes the form of a container with a round section and a flat bottom 10.

[0065] The insert has an internal cavity 11, which is delimited by a side wall 12 of the insert and the bottom 10.

[0066] The cell culture is carried out in the cavity 11 of the insert 1, to produce the cell tissue 2 which is shown in particular in figure 15.

[0067] The side wall 12 of the insert 1 has first through holes 13, which are small enough to prevent the passage of a cell culture fluid (in this example, it is a hydrogel) in which cells are capable of reproducing, when the cell culture fluid (the hydrogel in the example described) is above a predetermined temperature.

[0068] The hydrogel is the cell culture fluid chosen to feed the cells and to maintain the physiological conditions of the environment in which the cells will develop (for example pH, osmolarity, etc.). It can be supplemented with serum and antibiotics if necessary.

[0069] A "hydrogel" is a gel in which the swelling agent is water. The matrix of a hydrogel is generally a network of polymers. These are insoluble in water, but are capable of swelling substantially in the presence of large amounts of water or aqueous solutions such as biological fluids.

[0070] An example of a hydrogel may comprise 5% (wt / vol) bovine gelatin, 2% (wt / vol) very low viscosity alginate (Alpha Aesar #A18565) and 2% (wt / vol) fibrinogen (Sigma #F8630) dissolved in DMEM medium (Gibco™ #21068028) at 37°C.

[0071] In the context of our implementation example, the hydrogel presents:

[0072] - in the context of a first example of hydrogel (known under the name Bio ink, and whose composition is indicated in the previous paragraph) a viscosity which can be 1.4 x 10 5 Pa.s (or substantially 1.4 x 10 5Pa.s) at a temperature of 25 °C, preferably 3.3 x 10' 1 Pa.s (or substantially 3.3 x 10' 1 Pa.s) at 37°C, and

[0073] - in the context of a second example of Fibrinogen hydrogel (2% (weight / volume)), a viscosity which can be 4.7 x 10' 2 Pa.s (or substantially 4.7 x 10' 2 Pa.s) at a temperature of 25 °C, preferably 1.2 x 10' 2 Pa.s (or substantially 1.2 x 10' 2 Pa.s) at 37°C

[0074] In the context of the invention, the viscosity of the cell culture fluids may be greater than or equal to 0.69 mPa.s at 37°C (viscosity of water).

[0075] The diameter of the through holes 13 is between substantially 1000 and 4000 pm. They are also called “capillary holes”.

[0076] At room temperature, the hydrogel has a surface tension such that it fits into the through holes without passing through them: this is how the through holes 13 serve as anchor points for the hydrogel and the cells that develop in the hydrogel.

[0077] This allows the cell culture to at least partially insert into the through holes 13 to anchor therein, thereby allowing the created cell tissue to be retained on the side wall 12 of the insert 1.

[0078] The holes 13 are, in the context of the example shown, uniformly distributed all around the wall 12 so that the cell tissue created can anchor itself uniformly all around the wall 12 of the insert 1, and form a generally uniform pellet in the insert not retracted on itself, with regular dimensions in height and thickness (see figure 15).

[0079] It should be understood that the through holes 13 of the wall 12 of the insert 1 could have another shape and be more or less numerous without departing from the scope of the invention or distributed unevenly.

[0080] In the illustrated example, the through holes 13 are distributed around the side wall 12 equidistant from each other and form at least a first row of first through holes.

[0081] An insert with a wall 12 having several rows of through holes 13 could also be provided without departing from the scope of the invention.

[0082] Figures 1 and 2 also show that the bottom 10 of the insert 1 has second through holes 14.

[0083] These second through holes 14 allow the observation of the cells in culture through the bottom 10 of the insert 1, for example by means of a device equipped with a camera positioned under a transparent support, on which the insert or the container equipped with the insert rests.

[0084] The observer can also look from below the insert to observe the cell tissue through the second through holes 14. It is intended that the second through holes 14 are also capillary holes also capable of retaining the hydrogel in said cavity 11 and serving as an anchor for the cell culture.

[0085] The second through holes preferably have a diameter of between 1000 and 4000 μm. In this example, hole diameters of substantially 2000 μm (or substantially 2000 μm) are provided. Thus, the through holes 13 (hereinafter referred to as first through holes, to distinguish them clearly from the other through holes) and the second through holes 14 serve to anchor said cell tissue, the second through holes 14 of the bottom 10 of the insert also serving to observe the development of the cell culture and the cell tissue obtained.

[0086] It can be seen in Figures 1 to 3 that insert 1 has two side tabs

[0087] 15, projecting from the side wall 12 and which extend in a radial direction.

[0088] As can be seen in Figure 15, these tabs 15 can be used to grip the insert 1, for example with pliers.

[0089] The legs 15, however, have another main function: they allow the insert 1 to be positioned, in a removable manner, in the container 3 (the container 3 will be described later), or even at different levels (or positions) in the container 3. It is noted that each of the legs 15 has a transverse blind hole

[0090] 16, which also serves in the removable fixing of the insert 1 in the container 3, as will be seen later (second fixing element).

[0091] Figures 1 and 2 also illustrate that the side wall 12 of the insert 1 has a free end which forms a notch 17, on which four bosses 18 protrude.

[0092] It should be understood that other embodiments could be envisaged without departing from the scope of the invention and that the insert 1 could have more or fewer bosses 18: for example, a single projecting boss 18, two diametrically opposed projecting bosses 18, or three bosses distributed equidistantly or not, etc.

[0093] Other positioning means, other than bosses and blind holes, could be provided: elements of complementary shapes and capable of being assembled and disassembled could be provided without departing from the scope of the invention. In parallel, in the vicinity of the edge of the bottom 10 of the insert (see figure 2) which joins the side wall 12, the bottom 10 has four housings 19, of a shape complementary to that of the four bosses 18. In this way, the housings 19 of a first insert 1 can receive the bosses 18 of a second identical insert, positioned under the first insert. This makes it possible to stack several inserts on top of each other, so as not to lose them, to arrange and store them compactly (see figure 3). This also makes it possible to carry out several cultures simultaneously, in the same conditions it is thus possible to cultivate several identical or different tissues, at the same time, without bringing them into contact.

[0094] In Figure 3, we notice that the inserts 1 are stacked with their protruding legs 15 offset by 90 degrees. Another form of stacking could be envisaged: the legs 15 can also be aligned one above the other.

[0095] Reference will now be made to Figures 4 and 5 to describe the container 3 of the device according to the invention, capable of receiving the insert 1.

[0096] Container 3 is generally cylindrical in shape.

[0097] It comprises an internal chimney 30, delimiting an internal wall 31 of said container 3.

[0098] In the illustrated example, the chimney 30 is cylindrical in shape.

[0099] It should be understood that the shape of the chimney 30 could be different, without departing from the scope of the invention. The internal shape of the chimney is however complementary to the external shape to be able to accommodate it.

[0100] The container 3 has an external side wall 32, which surrounds the internal wall 31 formed by the chimney 30.

[0101] The inner wall 31 forms a conduit in the container 3, the conduit having a first opening 33 at a first end of the conduit (figure 4) and a second opening 34 at a second end of the conduit (figure 5), opening into a bottom 35 of the container 3.

[0102] A bottom wall 36 of container 3 connects the bottom ends of the inner 31 and outer 32 walls.

[0103] Thus, the bottom walls 36, internal 31 and external 32 and the bottom wall 36 form a tank 39 (annular in the context of our example) around the chimney. As indicated, the chimney 30 has internal dimensions larger or substantially larger than the external dimensions of the insert 1, and a shape complementary to the shape of the insert to be able to accommodate it. The first opening 33 allows the insertion of the insert 1 into said chimney 30 of the container 3, as illustrated in FIG. 6. The second opening 34 allows the observation of the cell culture through the insert 1, since the bottom of the insert 1 is visible through this opening 34.

[0104] Reference 4 represents the hydrogel in which the cell culture is carried out.

[0105] Two axial notches 37 are made through the internal wall 31, at least over part of the height of the chimney 30, to accommodate the projecting legs 15 of the insert 1.

[0106] It can be seen in particular in Figures 4 and 5 that the notches 37 have, on their edge, four projecting pins 38 which are positioned at four different levels in the notches 37.

[0107] The projecting pins 38 ensure the removable fixing of the insert 1 in the chimney 30, by insertion of the projecting pins 38 into the blind holes 16 of the legs 15 of the insert 1.

[0108] Thus, the insert 1 can be positioned in the chimney 30 at four different levels, for the reasons which will be explained later.

[0109] The protruding pins 38 are half-dome shaped and are deformable to fit into and out of the blind holes 16, which facilitates changing the position of the insert in the container 3.

[0110] It can be seen in Figures 4 and 5 that the internal wall 31 of the chimney 30 has third through holes 23 and 43, which are distributed over three rows: two rows of through holes 23, each round in shape, correspond to two levels of positioning of the insert in the container, these two levels being the lowest level (where the insert is positioned at the bottom of the container - see Figures 7 and 8) and the level called "low intermediate", located above the low level.

[0111] The row of through holes 43, each of axially oriented oblong shape, extends over two other levels of attachment of the insert in the container 3: the upper intermediate level, located above the lower intermediate level, and the upper level. The third through holes 23 and 43 are thus provided through the wall of the chimney 30 and align with the first through holes 13 of the wall 12 of the insert when the insert is positioned in the container, at the lower level, at the lower intermediate level, or upper intermediate level, and at the upper level.The alignment of the holes 13 and 23, or 13 and 43, allows the passage of the feed fluid introduced into the tank 39, through the wall 12 of the insert and through the internal wall 31 of the container, from the cavity 11 to the tank 39 or from the tank 39 to the cavity 11, when the insert is set in motion and when the viscosity of the hydrogel allows it: this is possible if the hydrogel is at a temperature lower than the predetermined temperature mentioned above.

[0112] Indeed, under the predetermined temperature (i.e. substantially under a temperature which may be between 15 and 15°C), the hydrogel has a lower viscosity, allowing it to have a lower surface tension, which allows the hydrogel to pass through the first through holes 13. The hydrogel does not pass through the through holes 14 when the bottom of the insert is positioned on a flat support, on which the bottom of the container 36 is also positioned and when the insert 1 is positioned in the container 3 at the low level.

[0113] Indeed, in accordance with the method according to the invention, it is planned to set in motion the device according to the invention, and therefore the container 3 receiving the insert 1, during the culture of the cells. This makes it possible to generate constraints on the cells and to recreate stress conditions allowing the cells to react and acquire mechanical properties close to those of real skin.

[0114] During movement of the device, the feed fluid flows through the first and third through holes, which further promotes anchoring of the tissue in the through holes 13 of the insert.

[0115] For easy passage of the feed fluid through the through holes 13 and 23 or 43, the holes are of the same shape and size.

[0116] The presence of an oblong shape allows for a greater passage of the feed fluid. It also allows the hydrogel to pass through several rows of first through holes 13. It should however be understood that the through holes 13, 23 and / or 43 could be of different shapes and sizes without departing from the scope of the invention.

[0117] When the insert is positioned in the container 3, it is possible to monitor the evolution of the generation of the cell tissue without having to remove the insert from the container: in fact, as explained previously, the opening 34 (second opening of the chimney 30) allows the insert 1 to be seen from below, and the second through holes allow the hydrogel and the cell culture to be viewed in the cavity of the insert.

[0118] Figures 7 and 8 illustrate the insert 1 positioned at the bottom level of the chimney 30 of the container 3: the through holes 13 of the side wall 12 of the insert 1 are positioned opposite the row of third holes 23 of the bottom level. The insertion of the tabs 15 of the insert 1 into the notches 37 of the chimney 30 imposes a direction of orientation (or a position) of the through holes 13 of the insert relative to the through holes 23 or 43 of the chimney. In addition, the fact that the tabs 15 are inserted into notches 37 prevents any rotation of the insert 1 relative to the container, which ensures that the first and third through holes remain well aligned.

[0119] In this position, where the insert is at the low level in the container 3. This position allows the production of gelled cell cultures.

[0120] Figures 9 and 10 illustrate the insert 1 positioned at the lower intermediate level of the chimney 30 of the container 3. This position makes it possible to carry out cell cultures in submersion.

[0121] Figures 11 and 12 illustrate the insert 1 positioned at the upper intermediate level of the chimney 30 of the container 3. This position makes it possible to carry out cell cultures with air / liquid contact.

[0122] Figures 13 and 14 illustrate the insert 1 positioned at the top of the chimney 30 of the container 3. This position makes it possible to produce tissues on which cosmetic tests can be carried out, in vitro drug screening, to study the toxicity of certain products, etc.

[0123] Being able to position the insert at several levels (at least two different levels) to create different cell development layers, makes it possible to reproduce the different layers of the skin and to introduce an air / liquid interface during the growth of the tissue (skin) if necessary. Figure 16 schematically represents an installation according to the invention, which comprises at least one device as described above (i.e. comprising at least one insert 1 and one container 3).

[0124] Figure 16 illustrates two devices each represented in such a way that the bottom 36 of the container 3 rests on a flat support 5 which is inclined relative to a horizontal plane H.

[0125] Two double arrows F schematically illustrate that the flat support 5 is driven in movement (it is mobile) by a movement drive device.

[0126] The motion training apparatus has not actually been shown in Figure 16 but is shown diagrammatically by the double arrows F.

[0127] The movements caused by the device are symbolized by the arrows F1, F2 and F3 illustrated above the devices in accordance with the invention.

[0128] The movement illustrated by arrow F1 corresponds to a pendulum movement: the moving drive device forces the flat support to pivot around a horizontal axis, which causes the support 5 to tilt (inclination) from one side to the other relative to the horizontal H, as illustrated in figure 16.

[0129] The movement symbolized by the arrow F2 is a rotational driving movement around a vertical axis: the support 5, on which the device according to the invention is positioned, can be rotated around its vertical axis.

[0130] Finally, the movement symbolized by the arrow F3 is a movement which combines rotation and pendulum.

[0131] Through the movements of the feed fluid and the entire device, physical constraint is applied to the cultured cells and stress is generated, which leads to a development of the cell culture close to that which actually occurs during the actual reproduction of cells in a living body.

[0132] The movement drive of the cell culture being developed, combined with positioning of the insert 1 in the container 3 on several levels (allowing different conditions for the development of the cell culture) allows the production of cell tissues in the insert that are more resistant than those produced by the devices of the prior art. The movement of the cell culture also allows better anchoring of the tissue in the first and second through holes 13 and 14 presented previously.

[0133] The development of the cell culture in the insert 1 can be observed using an optical device 6, as also illustrated in FIG. 16. The installation may comprise this device, positioned under the transparent plane support 5. The optical device 6 may also be independent and associated with the installation according to the invention.

[0134] Reference will now be made to a method for producing a tissue 2 of cells, as illustrated in Figure 15, by implementing the installation shown in Figure 16, comprising at least one device in accordance with the invention.

[0135] Firstly, the insert 1 is positioned at the lowest level in the chimney 30 of the container 3, and the container 3 is positioned on the flat support 5, so that the bottom 36 of the container and the bottom 10 of the insert 1 rest on the flat support 5.

[0136] Hydrogel is introduced into the insert 1, as well as cells to be cultured in the cavity of said insert, into the hydrogel.

[0137] It should be noted that, when the hydrogel is introduced into the insert 1, it is at a temperature lower than the temperature such that it has a viscosity allowing it to pass through the first, second, and third through holes (respectively 13, 14, 23 and 43).

[0138] Since the process is carried out at room temperature, the hydrogel thickens (increase in viscosity), and after a few seconds, the viscosity of the hydrogel is such that it no longer passes through the first, second and third through holes 13, 14, 23 and 43, thus allowing it to anchor there.

[0139] The motion drive apparatus of the installation is then put into operation and the support on which the device according to the invention rests is driven in movement for a predetermined time. The fluid is then driven into movement in the insert, generating stress on the hydrogel and the cells cultured therein, for said predetermined time: the nutrient fluid crushes on the insert and generates a force or stress on the hydrogel containing the cells. This has the effect of maturing the cells inside the hydrogel to produce more collagen protein. More generally, this has the effect of inducing either differentiation of the cells or production by the cells of specific extracellular matrix, or self-organization of the cells into functional microstructures (microvessels for example). These three phenomena (non-exhaustive) lead to the maturation of a functional tissue.A first layer of cell tissue is then obtained in the insert 1. During this first step, the development of the cells can be observed using the optical device 6, through the second through holes 14 at the bottom of the insert.

[0140] At the end of the first predetermined time, insert 1 is moved into container 3 to be positioned at the lower intermediate level (i.e. just above the lower level).

[0141] The support is then driven into motion by the motion drive device (movement F1, F2 or F3) for a second predetermined time.

[0142] This operation can be repeated by moving the insert from the low intermediate level to the high intermediate level, or to the high level, and the support can again be driven in movement (or not) for a predetermined time, until the cell tissue obtained conforms to what is sought: for example, certain steps can be repeated, the position of the insert in the container can be changed, the movement of the flat support 5 can be changed, or the driving apparatus can not be activated in movement and the cell tissue can be waited for to have developed within the insert 1 and to have a certain thickness (corresponding for example to the depth of the insert 1).

[0143] It is thus understood how the device according to the invention makes it possible to create cell tissues that are more resistant than those obtained by the devices of the state of the art by creating stress conditions during the cell development phases, by changing the positioning of the insert 1 in the container, and creating movements of the hydrogel in the insert 1, etc.

[0144] Thanks to the presence of the capillary holes 13, 14, the cell culture tissue is anchored in the walls of the insert, which makes it possible to obtain a tissue close to a tissue of real skin cells in reconstitution for example, pulling on the healthy edges of a wound, for example: we observe in fact in figure 15 that the surface of the tissue 2 forms a concavity oriented towards the outside of the insert. And makes it possible to obtain a tissue which has not retracted on itself. The tissue thus has properties even closer to the natural tissues which reconstitute themselves after an injury during the healing phase.

[0145] It should be understood that the invention is not limited to the embodiment shown in the figures and that it extends to the implementation of any equivalent means.

[0146] The device according to the invention may be made from different materials without departing from the scope of the invention, for example polyacrylate, polycarbonate, polystyrene, ceramic, stainless steel, polyethylene terephthalate.

Claims

CLAIMS 1. Cell culture device comprising an insert (1) having an internal cavity (11) delimited by an insert side wall (12) and an insert bottom (10), said internal cavity (11) being capable of receiving said cell culture in a cell culture fluid so as to create a cell tissue (2), said device comprising a container (3) in which said insert (1) is received in a removable manner, said insert side wall (12) having first through holes (13), characterized in that said container (3) comprises an internal chimney (30), delimiting an internal wall (31) of said container (3) and forming a conduit having a first opening (33) at a first conduit end and a second opening (34) at a second conduit end, opening into a container bottom (36), said first opening end (33) allowing the insertion of said insert (1) into said internal chimney (30) of the container (3),in that said insert bottom (10) comprises second through holes (14) allowing observation of the cells in culture through the insert bottom (10) by the second opening (34) of the second conduit end of said chimney (30), said first and second through holes (13, 14) being capillary holes capable of retaining said cell culture fluid in said internal cavity (11) of said insert and serving as anchoring to said cell tissue (2)., 2. Device according to claim 1, characterized in that said chimney (30) comprises third through holes (23, 43), arranged in the internal wall (31) which cooperate with said first through holes (13) when the insert (1) is received in said chimney (30), said third through holes (23, 43) ensuring the passage of a feed fluid between the cavity (11) of the insert and a tank (39) for receiving said feed fluid, said tank (39) being delimited between a side wall (32) of the container (3), the bottom (36) of the container (3) and the internal wall (31) formed by the chimney (30).

3. Device according to claim 2, characterized in that said insert side wall (12) comprises at least two rows of first through holes (13) and in that the internal chimney (30) comprises at least least two rows of third through holes (23, 43), at least one of said at least two rows of first through holes (13) cooperating with at least one of said at least two rows of said third through holes (23, 43) when said insert (1) is inserted into said chimney flue (30). Device according to claim 2 or 3, characterized in that said first through holes (13) and said second through holes (14) have substantially the same shape and the same dimensions. Device according to any one of the preceding claims, characterized in that said chimney (30) comprises first fixing elements (38) removable from the insert (1), the insert (1) being equipped with second removable fixing elements (16) which are associated with said first removable fixing elements (38).Device according to claim 5, characterized in that said first removable fixing elements (16) of the insert (1) are distributed over at least two levels in said chimney (30), each of said at least two levels being located between said first and second ends (33 and 34) of said chimney (30).Device according to claim 5 or 6, characterized in that said insert (1) comprises at least one external lateral projecting tab (15), said projecting tab (15) comprising at least one blind hole (16), in that said chimney (30) comprises a notch (17) arranged in an axial direction through the wall (31) of chimney (30), the notch (17) having an edge comprising at least one projecting pin (38), said at least one blind hole (16) of said at least one projecting tab (15) constituting said at least one first fixing element of the insert and said at least one projecting pin (38) of said chimney (30) constituting said at least one second fixing element of the chimney, associating removably with said at least one first fixing element of the insert.Device according to any one of the preceding claims, characterized in that said insert (1) comprises at least one boss (18) on an edge (17) formed by a free end of said side wall (12) of the insert, and at least one housing (19) provided in the vicinity of the edge of the bottom (10) of said insert (1), said at least one housing (19). having a shape complementary to that of said at least one boss (18) to receive said at least one boss (18) of a second additional and identical insert (1) positioned under the insert (1) of said device.

9. Insert (1) for a device according to any one of the preceding claims, comprising an internal cavity (11) delimited by an insert side wall (12) and an insert bottom (10), said cavity (11) being capable of receiving said cell culture in a cell culture fluid so as to create a cell tissue (2), said insert side wall having first through holes (13), characterized in that said insert bottom (10) comprises second through holes (14) allowing observation of the cells in culture through the bottom (10) of the insert, said first and second through holes (13, 14) being capillary holes capable of retaining said cell culture fluid in said internal cavity (11) of said insert (1) and serving as anchoring to said cell tissue (2).

10. Insert according to claim 9, characterized in that said first and second through holes (13, 14) have a diameter substantially between 1200 and 2000 μm.

11. Insert according to claim 9 or 10, characterized in that it comprises at least one boss (18) on an edge (17) formed by a free end of said side wall (12) of the insert, and at least one housing (19) provided in the vicinity of the edge of the bottom (10) of said insert, said at least one housing (19) having a shape complementary to that of said at least one boss (18) to receive said at least one boss (18) of a second additional and identical insert (1) positioned under the bottom (10) of the insert (1).

12. Installation comprising at least one device according to any one of claims 1 to 8, as well as a support (5) on which said at least one device can be positioned, said support (5) being mounted movably by being associated with a movement drive apparatus (F1, F2, F3, F), to drive said cell culture in movement in said insert (1) during the formation of said cell tissue (2).

13. Method for producing a cell tissue, using an installation according to claim 11, said method being characterized in that it comprises the following steps: a) positioning said insert (1) in said chimney (30) of said container (3) b) positioning said device on said support (5) of said installation, c) introducing a cell culture fluid and cells to be cultured into the cavity (12) of said insert (1), d) actuating the movement drive apparatus (F1, F2, F3, F) of said support (5), at least partially for a predetermined time, to cause a movement of said cell culture fluid in said insert (1) and of the cells in culture in said cell culture fluid, until at least a portion of cell tissue (2) is obtained in said insert (1), e) removing said device from said support (5), and f) extracting said insert (1) from said container (3), said insert (1) comprising said cell tissue (2).Method according to claim 13, said installation comprising an observation device (6) positioned under said support (5), said support (5) being transparent, characterized in that it comprises an additional step d1) between step d) and step e), according to which the development of said cell tissue (2) is observed through said second through holes (14) of said bottom (10) of the insert (1). Method according to claim 13 or 14, implementing an installation according to claim 11 comprising a device according to claim 7, the chimney (30) of said receiving container (3) comprising four projecting pins (38) of the axial edge (17) formed in the wall (31) of the chimney (30), to position said insert (1) at four different levels in said chimney (30) between the first and second openings (33, 34) of said chimney (30), the four projecting pins (38) comprising:. - a first projecting pin (38) substantially at the level of the bottom (36) of said container (3), in the vicinity of said second opening (34), said bottom level of said container defining a first level in said chimney (30), - second and third projecting pins (38) between said second opening (34) and said first opening (33) of the chimney (30), the positioning of the second and third pins (38) defining a second and third level of positioning of said insert (1) in the chimney (30) of the container (3), - a fourth projecting pin (38) substantially at the level of the first opening (34) of said chimney (30), the fourth pin (38) defining a fourth level of positioning of said insert (1) in the chimney (30), characterized in that, in step c), said insert (1) is positioned at said first level of said chimney (30), then optionally step d) is carried out or the cells are allowed to develop, for a first predetermined time, in that, at the end of said first predetermined time, the insert (1) is positioned at said second level, then optionally step d) is carried out again, in that, at the end of a second predetermined time, the insert (1) is positioned at said third level, then optionally step d) is carried out again, and in that, at the end of a third predetermined time, the insert (1) is positioned at said fourth level, then optionally step d) is carried out again, or the cells are allowed to develop,for a fourth predetermined time.,

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