WO2024068819A1

WO2024068819A1 - Device for cell or tissue culture

Info

Publication number: WO2024068819A1
Application number: PCT/EP2023/076847
Authority: WO
Inventors: Christophe Marquette; Hamza Raza ZAIDI
Original assignee: Université Claude Bernard Lyon 1; Centre National De La Recherche Scientifique; Institut National Des Sciences Appliquees De Lyon; Ecole Sup Chimie Phys Electroniq Lyon
Priority date: 2022-09-30
Filing date: 2023-09-28
Publication date: 2024-04-04
Also published as: FR3140377A1

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

DESCRIPTION

TITLE: DEVICE FOR CELL OR TISSUE CULTURE

FIELD OF INVENTION

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.

Cell culture and tissue culture are considered to be two types of processes used for the growth of cells of multicellular organisms outside their natural environment.

The goal of cell culture is to grow cells outside the body. Its use is important: it allows pharmacological and chromosomal studies to be carried out, it helps in fundamental research, the production of antibodies, protein synthesis, etc.

STATE OF THE ART

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

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

These skin equivalent models are increasingly in demand and created following the European Union's complete ban on testing on animals.

Equivalent skin models are created by culturing cells in devices, and are presented, for example, in the form of artificial skin tissue.

The methods used make it possible to create artificial skin tissue. The goal is to create artificial skin tissues, or artificial cells, that have similar properties to native human skin, both structurally and functionally. More generally, the objective is to create soft tissues, for example skin tissues but also liver, kidney, lung, adipose tissues, pancreas, etc.

A known way to produce these artificial skin tissues is to use cell culture inserts. These inserts are designed to enable static cell culture during which skin cells are matured without stressful environmental conditions, until obtaining the fabric of desired size and thickness.

The document WO2011127945 describes, for example, a cell culture system using inserts which are placed in a container and into which the cells or tissues are introduced to be cultured. Each insert makes it possible to create a tissue of cells which is subsequently used for testing 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, provided in a support. A liquid, allowing cell culture, is introduced into the wells.

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.

At the origin of the invention, we sought to improve the properties of the artificial skin obtained, that is to say to improve the properties of the tissue of cells so that it is even closer to a tissue of natural skin: in fact, until now, the artificial skin tissues created do not really imitate certain natural mechanical resistance properties that natural skin presents during its development.

It is known that the natural growth of human skin occurs simultaneously when the human body is exposed to multiple stresses and environmental conditions: It has been observed, from several studies, that when fibroblast cells are exposed to different types of stress, this leads to 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.

The production of collagen proteins provides some mechanical strength to the overall structure of the skin. It is these natural mechanical resistance properties that the invention aims to obtain in the artificial skin tissues created.

Some studies have succeeded in developing skin maturation bioreactors by inducing stress using multiple techniques. However, these developed bioreactors are very complex to build and are not accessible on the market. There is no dedicated bioreactor available on the market yet for dynamic maturation of skin tissues.

PRESENTATION OF THE INVENTION

The invention therefore aims to propose a device which 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 allowing better visual control (or by microscopy) of cell development while avoiding manipulation of the culture itself (in a non-destructive manner).

To this end, the invention relates 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 tissue of cells, said device comprising a container in which said insert is removably accommodated, said insert side wall having first through holes.

The device according to the invention is remarkable in that said container which accommodates the insert comprises an internal chimney, delimiting an internal wall to 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 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 through the second opening of the second conduit end 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 tissue of cells.

Indeed, and according to a first technical function that the invention allows, the through openings which are in the form of capillary holes serve as anchors for 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 you to see the bottom of the insert and the capillary through holes making it possible to observe the cell culture through the bottom of the insert without allowing the cell culture to “leak” through the bottom of the insert.

By “capillary” we will understand the fact 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.

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 there) to anchor in the first and second through holes that the insert presents, to allow the tissue of cells created to cling to the walls of the insert (side and bottom of the insert) in order to create a tissue which is distributed substantially homogeneously 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 surface initial cell tissue created, 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.

The device according to the invention may also comprise the following characteristics, taken separately or in combination: Advantageously, the chimney comprises third through holes, provided 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 feeder fluid between the cavity of the insert and a tank for receiving the feeder fluid, said tank being delimited between a side wall of the container, the bottom of the container and the wall internal formed by the chimney. This allows movement of the feeder fluid, in which the cell culture fluid is immersed (for example hydrogel in the context of an example which will be described and illustrated subsequently) which can be accommodated in the device during culture. cells, when the device is subjected to movement, to recreate stressful conditions allowing cells to react and acquire mechanical properties close to those of real skin.

According to a non-limiting embodiment described below, 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.

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 comprised (or substantially comprised) between 1000 and 4000 pm, preferably less than or equal to 2000 pm (or substantially 2000 pm).

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 insert is fixed in the chimney by means of removable fixing elements, the insert and the container being equipped with complementary means which, when combined, allow this removable fixing.

The first removable fixing 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. We can thus create layers of different cell development, to reproduce the layers of the skin and introduce an air/liquid interface during the growth of the tissue (skin) if necessary.

In another 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 formed in an axial direction through the chimney wall, the notch having a edge having at least one protruding pin, said at least one blind hole of said at least one protruding tab constituting said at least first fixing element of the insert and said at least one protruding pin of said chimney constituting said at least one second fixing element of the chimney, associating removably with said at least one first fixing element of the insert .

The protruding tab serves here as a removable fixing means. As will be seen later, it can also be used as a gripping element to grasp the insert. Preferably, the insert comprises at least one boss on a 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 several inserts of the device to be assembled one on top of the other by stacking.

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 tissue of cells, said side wall of insert having first through holes. The insert according to the invention is remarkable in that the bottom of the insert comprises second through holes allowing the observation of 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 an anchor for the cell tissue.

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

Furthermore, the insert comprises at least one boss on a 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.

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 movable while being associated with a moving drive device, to drive in movement the nourishing fluid in which the culture medium is immersed in said insert during the formation of said tissue of cells.

The invention finally relates to a method for producing a tissue of cells, 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 in the cavity of said insert, step c) being able to be carried out before step a) , d) introduction of a feeder fluid into said device, e) actuation of the device for driving said support in movement at least partially for a predetermined time, to cause movement of said feeder fluid in said insert, until obtaining of at least a portion of cell tissue in said insert, f) removing said device from said support, and g) extracting said insert from said container, said insert comprising said cell tissue.

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 tissue of cells is observed through said second through holes of said bottom of the insert.

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

- a first pin projecting 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,

- second and third projecting 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 positioning level of said insert in the chimney of the container,

- a fourth pin protruding 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 in said chimney first level of said chimney, then step d) is optionally carried out where 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 we carry out possibly repeat step d), after a second predetermined time, the insert is positioned at said third level, then step d) is optionally carried out again.

This step makes it possible to optionally introduce an air/liquid interface: the lower surface and the peripheral edge of the cell culture fluid are in contact with the culture medium (feeder fluid) while the upper surface of the insert is kept dry to the maturation of keratinocytes, for example. The upper oblong-shaped hole of one embodiment, which will be described subsequently, is intended to stop the flow of the feed fluid due to surface tension.

Finally, and at the end of a third predetermined time, the insert is positioned at said fourth level, then step d) is optionally carried out again, where the cells are allowed to develop, for a fourth predetermined time. This step can allow the application of chemical compounds to be tested on the skin after it has matured.

PRESENTATION OF FIGURES

Other advantages and characteristics of the invention will appear on examination of the detailed description of a mode of implementation in no way limiting, and of the appended drawings, in which:

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

[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,

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

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

[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,

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

[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,

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

[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,

[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

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

[Fig. 16] schematically illustrates an installation according to the invention, as well as certain steps of a method according to the invention for implementing the installation.

MODE OF REALIZATION

Figures 1 to 3 only represent a device insert 1 conforming to one embodiment of the invention.

The insert 1 has the function of accommodating a cell culture and serves as a support for the tissue of cells that is created there.

According to the invention, the device also comprises a container 3 in which the insert 1 is removably accommodated: the container 3 will be presented subsequently with reference to Figures 4 and 5.

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

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

The cell culture is carried out in the cavity 11 of the insert 1, to produce the tissue 2 of cells which is shown in particular in Figure 15.

The side wall 12 of the insert 1 has first through holes 13, which are sufficiently small to prevent the passage of a cell culture fluid (in the context of this example, it is a hydrogel) in which cells are able to reproduce when the cell culture fluid (the hydrogel in the context of the example described) is above a predetermined temperature.

The hydrogel here is the cell culture fluid chosen to supply 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.

By “hydrogel” we will understand a gel in which the swelling agent is water. The matrix of a hydrogel is generally a network of polymers. The latter are insoluble in water, but are capable of swelling substantially in the presence of a large quantity of water or aqueous solutions such as biological fluids.

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

In the context of our example embodiment, the hydrogel presents:

- 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 ⁵ Pa.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° This

- 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

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

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

At room temperature, the hydrogel has a surface tension such that it inserts into the through holes without, however, crossing them: this is how the through holes 13 serve as anchoring points for the hydrogel and the cells. which develop in the hydrogel.

This allows the cell culture to at least partially introduce itself 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.

The holes 13 are, in the context of the example shown, uniformly distributed all around the wall 12 so that the tissue of cells created can be anchored 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).

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 non-uniformly.

In the context of the example illustrated, 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.

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.

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

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 rests or the container fitted with the insert.

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

The second through holes preferably have a diameter of between 1000 and 4000 pm. In the context of this example, hole diameters of approximately 2000 pm (or approximately 2000 pm) are provided. Thus, the through holes 13 (called first through holes hereinafter, to clearly distinguish them 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 used for observing the development of the cell culture and the cell tissue obtained.

We notice in Figures 1 to 3 that the insert 1 has two side tabs

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

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

The tabs 15, however, have another main function: they make it possible to position, in a removable manner, the insert 1 in the container 3 (the container 3 will be described subsequently), or even at different levels (or positions) in the container 3 Note that each of the legs 15 has a transverse blind hole

16, which is also used in the removable fixing of the insert 1 in the container 3, as will be seen later (second fixing element).

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 project.

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 less bosses 18: for example, a single projecting boss 18, two projecting bosses 18 diametrically opposite, or three bosses distributed equidistantly or not, etc.

Other means of positioning, other than bosses and blind holes, could be provided: elements of complementary shapes and capable of assembling and disassembling could be provided without departing from the scope of the invention. In parallel, near 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 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 allows you to stack several inserts on top of each other, so as not to misplace them, and to arrange and store them compactly (see figure 3). This also makes it possible to carry out several cultures simultaneously, under the same conditions we can thus cultivate several tissues, identical or not, at the same time, without bringing them into contact.

In Figure 3, we note that the inserts 1 are stacked with their projecting legs 15 offset by 90 degrees. Another form of stacking could be considered: the legs 15 can also be aligned one above the other.

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.

The container 3 is generally cylindrical in shape.

It includes an internal chimney 30, delimiting an internal wall 31 to said container 3.

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

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 accommodate it.

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

The internal 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 container 3.

A bottom wall 36 of container 3 connects the bottom ends of the internal 31 and external 32 walls.

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 significantly 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 insert 1 to be inserted into said chimney 30 of the container 3, as illustrated in Figure 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.

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

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 protruding legs 15 of the insert 1.

We note in particular in Figures 4 and 5 that the notches 37 have, on their edges, four projecting pins 38 which are positioned at four different levels in the notches 37.

The protruding pins 38 ensure the removable fixing of the insert 1 in the chimney 30, by inserting the protruding pins 38 into the blind holes 16 of the legs 15 of the insert 1.

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

The protruding pins 38 are in the shape of a half-dome and are deformable to insert and disengage from the blind holes 16, which facilitates changing the position of the insert in the container 3.

We note in Figures 4 and 5 that the internal wall 31 of the chimney 30 comprises third through holes 23 and 43, which are distributed over three rows: two rows of through holes 23, each of round 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 from the low level.

The row of through holes 43, each of oblong shape oriented axially, extends over two other levels of attachment of the insert in the container 3: the high intermediate level, located above the low intermediate level, and the high 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 low level, at the low intermediate level, or high intermediate level, and at the high level. The alignment of 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 towards the tank 39 or from the tank 39 towards 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 previously.

Indeed, under the predetermined temperature (that is to say substantially under a temperature which can 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 and when the insert 1 is positioned in the container 3 at the low level.

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

During the movement of the device, the nourishing fluid circulates through the first and third through holes, which further promotes the anchoring of the tissue in the through holes 13 of the insert.

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

The presence of an oblong shape allows greater passage of the nourishing fluid. It also allows the passage of the hydrogel 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 shape and size without departing from the scope of the invention.

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 visualization of the hydrogel and the cell culture in the cavity of the insert.

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 legs 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 guarantees that the first and third through holes remain well aligned.

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

Figures 9 and 10 illustrate the insert 1 positioned at the intermediate low level of the chimney 30 of the container 3. This position makes it possible to carry out submerged cell cultures.

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

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

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 introduce an air/liquid interface during tissue growth ( skin) if necessary. Figure 16 represents, schematically, an installation according to the invention, which comprises at least one device as described above (that is to say comprising at least one insert 1 and a container 3).

Figure 16 illustrates two devices each represented so that the bottom 36 of the container 3 rests on a plane support 5 which is inclined relative to a horizontal plane H.

Two double arrows F schematically illustrate that the plane support 5 is driven in movement (it is mobile) by a moving drive device.

The moving training device has not actually been shown in Figure 16 but is shown schematically by the double arrows F.

The movements caused by the device are symbolized by the arrows F1, F2 and F3 illustrated above the devices conforming to the invention.

The movement illustrated by the arrow F1 corresponds to a pendulum movement: the moving training device requires the plane support to pivot around a horizontal axis, which causes the support 5 to lean (tilt) to one side and of another in relation to the horizontal H, as illustrated in figure 16.

The movement symbolized by the arrow F2 is a rotational drive movement around a vertical axis: the support 5, on which the device according to the invention is positioned, can be driven in rotation around its vertical axis.

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

Thanks to the movements of the feeder fluid and the entire device, a physical constraint is applied to the cells in culture and a 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.

The movement of the cell culture during development, combined with positioning on several levels of the insert 1 in the container 3 (allowing different conditions for development of the cell culture) allows the production of cell tissues in the insert more resistant than those produced by state-of-the-art devices. The movement of cell culture also allows better anchoring of the fabric in the first and second through holes 13 and 14 presented previously.

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

Reference will now be made to a method making it possible to produce a tissue 2 of cells, as illustrated in Figure 15, thanks to the implementation of the installation shown in Figure 16, comprising at least one device conforming to the invention.

Initially, 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 The insert 1 rests on the flat support 5.

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

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 holes. through (respectively 13, 14, 23 and 43).

The process taking place 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 itself there.

The movement drive device 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 moved in the insert, generating stress on the hydrogel and the cells cultured therein, for said predetermined time: the nourishing fluid crashes onto the insert and generates a force or stress on the hydrogel containing the cells. This causes the cells inside the hydrogel to mature to produce more collagen protein. More generally, this has the effect of inducing either cell differentiation or production by cells of specific extracellular matrix, i.e. a self-organization of cells into functional microstructures (microvessels for example). These three phenomena (non-exhaustive) lead to the maturation of a functional tissue. We then obtain a first layer of cell tissue in the insert 1. During this first step, we can observe the development of the cells thanks to the optical device 6, through the second through holes 14 at the bottom of the insert.

At the end of the first predetermined time, the insert 1 is moved into the container 3 to be positioned at the low intermediate level (that is to say just above the low level).

The support is then driven in movement using the movement training device (movement F1, F2 or F3) for a second predetermined time.

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 is consistent with what is sought: for example, we can repeat certain steps, change the position of the insert in the container, change the movement of the plane support 5, or not train the device to activate training in movement and wait until the cell tissue has developed within the insert 1 and has a certain thickness (corresponding for example to the depth of the insert 1).

We thus understand how the device according to the invention makes it possible to create cell tissues more resistant than those obtained by the devices of the state of the art by creating stress conditions during the development phases of the cells, by changing the positioning the insert 1 in the container, and creating movements of the hydrogel in the insert 1 etc.

Thanks to the presence of 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 indeed observe in Figure 15 that the surface of the tissue 2 forms a concavity oriented towards the outside of the insert. And allows you to obtain a fabric that has not retracted on itself. The tissue thus presents properties even closer to natural tissues which are reconstituted after an injury during the healing phase.

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.

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

Claims

1. Cell culture device comprising an insert (1) having an internal cavity (11) delimited by a side wall (12) of the insert and an insert bottom (10), said internal cavity (11) being capable of receiving said culture cell in a cell culture fluid so as to create a tissue of cells (2), said device comprising a container (3) in which said insert (1) is removably received, 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) to said container (3) and forming a conduit having a first opening (33) at a first end of conduit and a second opening (34) at a second end of conduit, opening into a bottom (36) of container, 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 base (10) comprises second through holes (14) allowing the observation of cells in culture through the insert base (10) through 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 anchoring said cell tissue (2).

2. Device according to claim 1, characterized in that said chimney (30) comprises third through holes (23, 43), formed 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 feeder fluid between the cavity (1 1) of the insert and a tank (39) for receiving said fluid feeder, 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 at 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 fixing elements (16) which associate 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) formed in an axial direction through the wall (31) of the chimney (30), the notch (17) having a edge comprising at least one projecting pin (38), said at least one blind hole (16) of said at least one protruding tab (15) constituting said at least first insert fixing element and said at least one projecting pin (38) of said chimney (30) constituting said at least one second element for fixing 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 a edge (17) formed by a free end of said insert side wall (12), and at least one housing (19) provided in the vicinity of the bottom edge (10) of said insert (1), said at least one housing (19) having a shape complementary to that of said at least one boss (18) for receiving 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 a side wall (12) of the insert and a bottom (10) of the insert, said cavity (11) being capable of receiving said cell culture in a cell culture fluid so as to create a tissue of cells (2), said insert side wall having first through holes (13), characterized in that said insert bottom ( 10) comprises second through holes (14) allowing the observation of 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 fluid of cell culture in said internal cavity (11) of said insert (1) and serving as anchor 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 pm.

11. Insert according to claim 9 or 10, characterized in that it comprises at least one boss (18) on a edge (17) formed by a free end of said insert side wall (12), 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) for receiving 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 movably mounted being associated with a movement training device (F1, F2, F3, F), for driving said cell culture in movement in said insert (1) during the formation of said tissue of cells (2).

13. Method for producing a tissue of cells, implementing an installation according to claim 11, said method being characterized in that it comprises the following steps: a) positioning of said insert (1) in said chimney (30) of said container (3) b) positioning of said device on said support (5) of said installation, c) introduction of a cell culture fluid and cells to be cultured in the cavity (12) of said insert (1), d) actuation of the movement training device (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 cultured in said cell culture fluid, until at least a portion of cell tissue (2) in said insert (1), e) removal of said device from said support (5), and f) extraction of 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 tissue of cells (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 reception 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 the second opening (33, 34) of said chimney (30 ), the four projecting pawns (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 positioning level 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 positioning level 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 step d) is optionally carried out where 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 step d) is optionally carried out again, in that, at the end of a second predetermined time, we position the insert (1) at said third level, then we possibly carry out step d again), and in that, at the end of a third predetermined time, we position the insert (1 ) at said fourth level, then step d) is optionally carried out again, where the cells are allowed to develop, for a fourth predetermined time.