EP1280888A1

EP1280888A1 - Cell culture method, cell cluster obtained by said methods and use thereof

Info

Publication number: EP1280888A1
Application number: EP01927982A
Authority: EP
Inventors: Alain Othon André MILLER; Nathalie Liliane Paule Ghislaine Ropson-Kenda; Christophe Alexandre Maurice Soler
Original assignee: 4C Biotech SA
Current assignee: La Region Wallonne
Priority date: 2000-04-18
Filing date: 2001-04-17
Publication date: 2003-02-05
Also published as: FR2807765A1; AU2001254864A1; FR2807765B1; WO2001079453A1

Abstract

The invention concerns methods for obtaining anchor-dependent cells (CAD), by culturing said cells on two-dimensional micro-substrates (2D-MS2). The invention also concerns sheets, spheroids, organoids or cell aggregate, obtainable by said methods, and their uses, in particular for preparing artificial tissues or organs.

Description

Cell culture methods, cell clusters obtained by these methods and use thereof

The present invention relates to novel methods of culturing anchor-dependent cells on microsupports with two-dimensional cryosensitive geometry. More specifically, the invention relates to methods for obtaining anchored-dependent animal cells, individualized or forming cellular associations of leaf types, aggregates, spheroids or more organized organoids, having particular properties such as high cell viability. and homogeneity in size of the cell clusters.

The biotechnology industry is largely based on the cultivation of cells in vitro in order to generate and isolate certain biochemical components. Cultured cells are often genetically engineered to produce new molecules or molecules that are extremely difficult to isolate from organs or physiological fluids. The techniques for culturing prokaryotic cells and anchor-independent eukaryotic cells are well mastered by the industry. However, many cell lines and many types of normal anchor-dependent cells are used in industry for the production of biological, pharmaceutical or medical substances. Anchor-dependent cells (CAD) must imperatively physically interact with a support to be viable, functional and proliferate. This dependence constitutes a limiting factor in the culture of these cells compared to anchor-independent cells which, themselves, can proliferate in suspension.

More recently, therefore, techniques for mass culture of anchor-dependent eukaryotic cells have been exploited. To do this, several types of microcarriers are used industrially for the production of biological and pharmaceutical products, the best known of which are the three-dimensional Cytodex® (3D) microbeads from Pharmacia (UPPSALA, Sweden).

A new generation of microcarriers, with two-dimensional geometry, has been developed and described in EP 579 596.

By two-dimensional geometry, it should be understood that the thickness of these microcarriers tends to become minute and negligible compared to the dimensions of the cultured cells. This reduction in thickness is such that there is no possibility of cell growth, either within the support or on its edge, but only on its two anchoring faces. These 2D microsupports (2D-MS) have a high volume surface (approximately 950 cm ² / cm ³ ), and therefore offer the main advantage of an anchoring surface per unit of volume higher than all 3D competitors, such as the CYTODEX® microbeads mentioned above.

They therefore make it possible, for an occupation of culture volume given in a bioreactor, to cultivate and obtain more cells per unit of volume. Indeed, the external anchoring surface of a sphere (4πR ² ) is equal to the total surface of two infinitely thin discs (2 x 2πR ² ) located at the equator and forming part of this sphere. However, the combined volumes of these two "equatorial" discs become smaller the smaller the thickness of the film used to produce them, representing only a tiny fraction of the volume of the sphere which circumscribes them. Adopting a thickness of 10 μm for all of the disks generated in a sphere, the total surface capable of anchoring the cells thus provided by the disks is approximately 3.3 times greater than the outer surface of the sphere, taking into account the random movements of these, in suspension.

The large anchoring surface available for CAD per unit of volume 2D-MS therefore makes it possible to envisage the cultivation of cells at high concentrations on an industrial scale.

Another limitation of the supports for anchoring-dependent cell culture is that of the mode of recovery of the cells attached to their support, while preserving the biological or physiological properties of the latter. Indeed, the detachment of cells from their support goes through an enzymatic treatment (such as trypsin) or a chelating agent (such as EDTA), which can damage not only the cellular functions, but also their subsequent re-attachment to supports as part of continuous cultures. This limitation is particularly detrimental when the biological functions of the cells are then used industrially. This situation is frequently encountered in the case of production of macromolecules of interest by said cells, or even when all of the receptors or membrane molecules are sought for their capacity to bind ligands or internalize molecules or substances. The methods described above are therefore not adapted, qualitatively or quantitatively, to the cultivation of cells on an industrial scale with a view to producing large quantities of molecules with high added value such as glycoproteins sensitive to trypsinization of cells.

In addition, culture at the confluence of anchor-dependent cells leads to the formation of intercellular links or intercellular junctions. These intercellular junctions also contribute to the functionality of the cells and their destruction at the loss of these functionalities.

These limits in the mass culture of anchor-dependent cells also lead to a limit to the industrial production of biological macromolecules produced by said cells, whether these are molecules normally synthesized by the cells in question or more generally produced by insertion. by techniques of genetic recombination of genes encoding a heterologous protein. This limitation linked to the production capacities of functional anchoring-dependent cells can lead to industrial cost prices of the proteins which it is desired to express and purify, incompatible with a subsequent sale price of a medicament containing said protein as active ingredient.

Attempts to overcome a number of drawbacks described above, including the need to use enzymes or chelating agents to unhook cells from their support, have been described in EP 387 975 and in EP 382 214. These two patents propose to cover conventional cell culture supports with the product of the copolymerization of hydrophilic monomers chosen from poly-N-alkyl- (meth) acrylamide derivatives, their respective copolymers, poly-N-acryloyl-piperidine or poly -N-acryloyl-pyrrolidine whose desired functionality is cryosensitivity. The term “cryosensitive” indicates that the lowering of the temperature of a culture of cells attached to these 2D microcarriers, below a given temperature, leads to the detachment of these cells.

In this use, the polymer is chosen as a function of the lower critical solution temperature, or LCST, which is a transition temperature for the hydration and dehydration of the polymeric compound. When the LCST is lower than the cell culture temperature, the cells remain fixed on the polymer support during the cell culture phase. They can be detached from it following a drop in the temperature of the culture such that the latter is substantially lower than the LCST. The methods for obtaining a polymer or a copolymer with a given LCST temperature are described in EP 382,214 B1. In general, it can be said that the inclusion of hydrophilic monomers in the polymerization process tends to increase the LCST, while the presence of hydrophobic polymers tend to decrease the LCST. Examples of hydrophilic monomers are: N-vinyl-pyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methyl-acrylamide, hydroxyethyl-methacrylate, hydroxyethyl-acrylate, hydroxymethyl-metha-crylate, hydroxymethyl-acrylate, acrylic acid and methacrylic acid having acid groups and their salts, vinyisuifonic acid, styrylsulfonic acid and N, N'-dimethylamino-ethyl-methacrylate, N, N'-diethylamino-ethyl-methacrylate, and N, N'-dimethylamino-propyl-acrylamide having basic groups and their salts.

Examples of hydrophobic monomers are: acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methylmethacrylate and glycidyl methacrylate etc., N-substituted-aikyl- (meth) -acrylamide derivatives such as N -nbutyl- (meth) -acrylamide and N-isopropylacrylamide etc. as well as vinyl chloride, acrylonitrile, styrene, and vinyl acetate etc.

However, the means described in these applications for coupling these cryosensitive polymers or copolymers on the cell culture supports do not allow:

"nor control of the quantity and therefore of the thickness of the grafted polymers, a particularly important parameter when it is desired to control the density of cells in culture;

"or to ensure a covalent coupling of the polymer or copolymer on the culture support; this is a major drawback insofar as it does not allow long-term conservation of these supports nor their reuse.

In a recent European patent application (No. 99402710.0), A. Miller et al. have described new cryosensitive microsupports with two-dimensional geometry (2D-MS2), onto which are grafted molecules conferring on the microsupport special properties such as cryosensitivity or specific adhesion. The originality of this application N ° 99402710.0 lies in particular in the production process of said 2D-MS2, which allows a covalent grafting of molecules whose functionality is sought. This process notably comprises the following succession of steps:

• continuous production of reels of polymer film with a thickness less than or equal to 35 μ from granules of a given polymer;

• activation of said polymer film by any means enabling reactive groups to be generated, in particular radicals or peroxide, hydroperoxide or amino functions;

• a covalent grafting between the activated polymer film and a polymer, a copolymer or a macromolecule of interest whose functionality is sought, said grafting being obtained by immersion of the film in a monomer solution, the copolymerization being initiated by the free radicals created by activation under β radiation, the immersion time being directly correlated to the desired thickness of the polymer grafted on the film; • if necessary, washing to remove the monomers not consumed and not fixed on the film;

• cutting of the polymer film by a process chosen according to the geometry and the size of the 2D microsupports sought; ^“ Where appropriate, a sterilization step, either by autoclaving or by radiation, the sterilization method being of course chosen as a function of the material to be sterilized.

When the nature of the polymer used for the production of the film polymer is of the aliphatic polyester type, the film is in essence bioresorbable / biodegradable and, in this case, it can be used as an implant in the living organism if the polymer used is of biomedical grade, preferably recognized by the FDA.

For essentially economic reasons, industrialists tend to transform the discontinuous process of culturing anchor-dependent cells into a semi-continuous process such as serial culture, a process in which the same microsupports are always reused, the inoculum. from part of the cells of the previous cycle.

One of the aims of the present invention is to meet the need expressed by manufacturers to obtain large quantities of intact individualized cells, in particular by using cryosensitive 2D microcarriers of the same type as those described in the application cited above.

The claimed invention therefore relates to methods of obtaining anchor-dependent cells (CAD), comprising the following steps: a) inoculation of cryosensitive microsupports with two-dimensional geometry (2D-MS2) by CAD, b) culture at a temperature higher than the lower critical temperature (LCST) of the polymer responsible for the microsupport's cryosensitivity, c) detaching the cells from the microcarriers, by lowering the temperature of the culture to a temperature substantially lower than said LCST, d) if necessary, reproduction steps b) and c) until the desired quantity of cells is obtained. Preferably, the microsupports seeded in step a) consist of a polymer film on which are grafted covalently the molecules ensuring the cryosensitivity of said microsupports. More preferably, the 2D-MS2s used are those described in European patent application No. 99402710.0.

In a first embodiment of the invention, the anchoring-dependent cells are obtained individualized or not, intact, on a large scale, as is illustrated in example 1. Cryosensitive microsupports with two-dimensional geometry (noted below 2D-MS2 ) are seeded by anchor-dependent cells and the culture continued at a temperature compatible with cell culture and higher than the LCST of the polymers grafted on the microcarriers, preferably at 37 ° C. The culture is continued until a stage preceding that of confluence, during which junctions weld the cells to each other. Preferably, to obtain the best possible yield, the culture is continued until the stage immediately preceding that of confluence. At this time, without stopping the agitation, the temperature of the culture is lowered and maintained at a temperature substantially lower than the LCST, until all the cells have become detached and float in the suspension in individualized form or aggregates. A temperature is considered to be “substantially lower than the LCST” if it is at least 25 ° C lower than the LCST. Preferably, the temperature is maintained at 4-10 ° C to ensure the detachment of the cells. The sedimentation of the cryosensitive 2D microcarriers, following the stopping of the agitation, makes it possible to cream the intact cells present in the supernatant. If necessary, part or all of these cells can be used to re-seed 2D-MS2s and complete a new cell amplification cycle.

In this embodiment of the invention, the cells used are preferably cells which do not form junctions very strong intercellular cells such as human dermal fibroblasts, CHOs (Chinese Hamster Ovary), murine fibroblasts L929, murine fibroblasts 3T3. However, the culture of epithelial cells such as keratinocytes can also be envisaged in medium without serum and at low calcium concentration (<0.1 mM). Thus, the harvested cells, whether individualized or not, keep their membrane proteins and in particular the extracellular segment thereof intact. These cells are of particular interest to scientists whose research requires the availability of intact membrane proteins, manufacturers who develop diagnostic, toxicology, screening and other tests using such proteins, as well as the pharmaceutical industry which uses such proteins for example in vaccine design.

A second embodiment of the invention makes it possible to reuse the 2D-MS2 after having unhooked the cells from it, and to automatically operate their reseeding so as to carry out what is known as serial culture, or continuous culture. In such a culture process, intact individualized cells are produced in each cycle, as illustrated in example 2. A first way of carrying out a culture in continuous is to harvest only a part of the unhooked cells (in general, more 90%, but this may vary depending on the cells cultivated), before carrying out a new culture cycle at 37 ° C. with stirring. A preferred approach for performing a serial culture is to not unhook all the cells from the microcarriers. To do this, it suffices to use 2D-MS2 in the process, a certain percentage (for example 2-10%) of the surface of each microsupport is not cryosensitive, thus conferring partial cryosensitivity (partial detachment). In this case, step d) of the method described above is carried out by resuspension and at culture temperature of the 2D-MS, after partial detachment in accordance with step c). So, proceeding from the as described in Example 1 and following the lowering of the temperature from 37 ° C to 4-10 ° C, 2 to 10% of the cells do not detach from the microcarrier. These are the cells that serve as the inoculum for the next cycle. The percentages given here are illustrative and not limiting. Any percentage between 2 and 80% can be used in the serial culture process. Partial detachment of the microsupport cells in step c) can also be achieved by reducing the thickness of the cryosensitive polymer deposit. For example, for a detachment of 90% of CHO cells, the thickness of the polymer can be reduced from 10 to 2 nanometers. The cells used in these culture methods are preferably cells whose intercellular junctions are weak or nonexistent, in any case in the type of culture medium used to cultivate them. Just as for the first embodiment of the invention, the cells produced here continuously by their ease of obtaining, interest the academic, pharmaceutical and biotechnological laboratories for the production yield, associated with the superior quality of the membrane proteins obtained.

In contrast to the individualized cells obtained by the methods described above, obtaining three-dimensional structures of homogeneous size, called, depending on the number of cell types that compose them, spheroids (cells all belonging to the same cell type), organoids ( or pseudo-organs made up of cells from different cell types) or aggregates (cell clusters characterized by heterodispersity in size, whether or not composed of several cell types), has many advantages, and the process according to the invention allows controlled production of these different types of cell clusters.

Fundamental investigation, large-scale screening of molecules of therapeutic interest (high throughput screening), toxicology, restorative medicine, and gene therapy in particular, constitute as many areas where cell clusters (organoids, spheroids, aggregates) find their full use. This is not the case, or to a lesser degree, for these same individualized cells which are not or only slightly adapted to the desired function.

The cells of the organoids (and spheroids and aggregates) are arranged together in such a way by the scaffolding that constitute the molecules of the extracellular matrix that both their viability and their physiological functions are improved, or even restored to the point of equal the performance of the intact organ (Koide et al., 1989; Matsushita et al., 1991; Matsushita et al., 1992). The physiological functions of the differentiated cells organized into organoids are maintained at a high level for longer periods than during their culture in monolayer (Tong et al., 1990; Tong et al., 1992).

The interest represented by three-dimensional cellular structures (aggregates, spheroids, organoids) has stimulated the development of several methods for their manufacture. Most of these methods consist in cultivating individualized cells in stationary or agitated culture so that the cells which are well individualized at the start eventually aggregate and form cellular aggregates. Certain methods use non-adherent substrates such as proteoglycans, polylysine,

PVLA (poly- / V-p-vinylbenzyl-D-lactonamide) and polyurethane foam (Ijima et al., 1997). But with all these methods, it is impossible to control the size of the cellular clusters formed and the size distribution of the spheroids, organoids or aggregates obtained is then very wide.

The viability and the functionality of the cells composing the three-dimensional cellular structures are dependent in particular on the size reached by these. Furthermore, from one cell line to another, the size of the cell clusters can vary (Carvalhal et al., 1997). For example, fibroblast spheroids can reach 150 to 500 μm in diameter. Preferably, the size of the 2D microcarriers used in the processes for obtaining CAD of the invention is chosen according to the cell type of the cultured cells, so as to obtain clusters of viable cells, without risk of anoxia of certain between them.

Other methods have been developed in order to be able to produce cellular clusters. These are methods using a mixture of cryosensitive polymer, poly-N-isopropyl acrylamide (PNIPAAm), and collagen as a cell culture support. In fact, PNIPAAm is an insoluble polymer above 32 ° C and dissolves below this temperature. Culture surfaces covered with this polymer therefore acquire the property of cryosensitivity (US patent No. 5,284,766). To do this, this mixture is placed at the bottom of conventional culture dishes and allowed to polymerize and dry. The cells are then seeded on this support and cultivated until confluence and formation of intercellular junctions. At 37 ° C, the PNIPAAm molecules have a certain hydrophobic character and the cells remain perfectly anchored on their support. By lowering the temperature of the culture, the cells can be detached from the support in the form of a single or multicellular sheet. Indeed, below 32 ° C, the PNIPAAm acquires a certain hydrophilic character and the cells detach from the support in the form of isolated cells and / or sheets (Takezawa et al., 1990; Takezawa et al., 1992; Takezawa et al., 1993). However, this technique has significant intrinsic limitations. It does not make it possible to manufacture large quantities of flakes or to make flakes of predetermined and uniform size.

It can therefore be seen that there is a need to overcome these problems of producing cell clusters of micrometric and homogeneous size. One of the aims of the present invention is to meet this need. More specifically, a third embodiment of the invention relates to new methods of culturing anchor-dependent cells on microcarriers with two-dimensional geometry (2D-MS), cryosensitive (2D-MS2), in order to generate coherent two-dimensional or three-dimensional structures, of homogeneous size, made up of cells belonging to the same cell line or not, thus achieving the culture of anchor-dependent cells in the form of cell sheets, spheroids or organoids in suspension. Thus, in this embodiment of the method of the invention, the culture step b) is continued until the confluence stage, and the CADs are obtained after step c) in the form of cell sheets. In a preferred implementation of this method of the invention, the size of the cell sheets obtained after step c) is controlled. This size essentially depends on the size of the microcarriers.

The re-culture, with suitable stirring, of these cell sheets, preceded or followed by the elimination of 2D-MS from the culture medium, makes it possible to obtain spheroids (if the sheets consist of a single cell type), or organoids (if the sheets have more than one cell type). If the cultivation step b) is continued beyond the confluence stage, the CADs are obtained after step c) in the form of cell aggregates.

Cryosensitive microsupports with two-dimensional geometry (2D-MS2) are seeded by anchor-dependent cells originating from the same cell line or not, and the culture continued at a temperature above the LCST, preferably at 37 ° C., up to at the confluence. The confluence stage is characterized by the fact that the cell monolayer is made up of cells welded together by strong junctions (desmosomes, tight junctions, etc.). At this time, without stopping the agitation, the temperature of the culture is lowered and maintained at a temperature substantially lower than the LCST, preferably at 4-10 ° C., until all the sheets are released. The 2D-MS2 sedimentation following the stopping of the agitation makes it possible to recover the cell sheets present in the supernatant. These slips, resuspended in culture medium at 37 ° C. spontaneously form spheroids or organoids in a few hours, even a few days. Alternatively, following the thermal detachment of the sheets, the temperature of the culture can be reduced to 37 ° C. and the spheroids or organoids then separated from the 2D-MS2.

The processes for obtaining cell sheets and spheroids in accordance with the invention are shown diagrammatically in FIG. 1.

Unlike the cells used in the first two embodiments of the invention, the cells used in this third embodiment of the invention preferably have a strong ability to establish strong intercellular junctions (desmosomes, adherent junctions, etc.). Epithelial cells such as pancreatic islet cells of Langerhans which secrete insulin, porcine, rat or human liver cells and human keratinocytes are used in a preferred embodiment of the invention. These spheroids or organoids can be used as models during pharmaco-toxicological studies, for the screening of pharmaceutical molecules, or even for diagnosis. They can also be used for the production of molecules of pharmaceutical and cosmetic interest, as is illustrated in examples 5a and b. This invention also makes it possible, with these cells, to mass produce functional organoids of homogeneous size which can thus serve as raw material for the production of organs for transplantation in diabetics, as described in Example 5c.

The invention therefore relates to a process for obtaining CAD in the form of cellular sheets, comprising the following steps: a) seeding cryosensitive microsupports with two-dimensional geometry (2D-MS) by CAD, b) culture at a temperature above the critical lower temperature (LCST) of the polymer responsible for the microsuppression cryosensitivity, up to the confluence stage, c) detachment of the cells from the microcarriers, by lowering the temperature of the culture to a temperature substantially lower than said LCST.

In a variant of this process, the microcarriers seeded in a) by CAD are previously covered with a "sublayer" of cells, preferably of a type different from that of said CAD. This “sublayer”, or primary layer, is, if necessary, treated at confluence by a physical process such as γ irradiation, or chemical such as mitomycin c, so as to stop their proliferation without altering the membrane functionalities. An example of this variant is described in Example 4.

The invention also relates to processes for obtaining spheroids or organoids, comprising the following steps: a) recovery of cell sheets obtained according to a process as described above, b) resuspension of these sheets in culture medium at 37 ° C, c) removal of 2D-MS from the culture medium.

If necessary, step b) above can be carried out before step c) during the production of the spheroids or organoids.

In this process, the cell sheets recovered in step a) are preferably homodisperse in size, and the size of the spheroids or organoids obtained is controlled. This size essentially depends on the size of the microcarriers, and on the morphology of the cultured cells. In another embodiment of the invention, organoids can be produced by a method comprising the following steps: a) obtaining spheroids or organoids by a method as described above, b) inoculating these spheroids or organoids with other

CAD, of the same cell type, or, preferably, of a different cell type, c) the culture is then continued until the surface of the starting spheroids or organoids is covered by the cells seeded in b), d) if necessary, steps b) and c) can be repeated to obtain organoids having a structure organized in “multiple layers”, or until the organoids have the expected size and composition.

An example of production of organoids by this process is presented in Example 5b.

Of course, the cell sheets, spheroids, or organoids capable of being obtained by any of the methods described in this application, fall as such within the scope of the present invention.

These sheets, spheroids or cell organoids have the following characteristics: they are of homogeneous size, linked to the size of the microcarriers, and are made up of cells which are for the most part viable and have an intact membrane. In fact, the processes for producing sheets, spheroids or cellular organoids described above allow good oxygenation and feeding of the cells, which are all in direct contact with the culture medium during most of their growth, ie that is to say until they drop the microcarriers. The processes for obtaining sheets, spheroids, or cellular organoids described above also make it possible to control the size of these structures. The use of microsupport of a given diameter will make it possible to obtain cell sheets of the same diameter, and spheroids or organoids of smaller diameter. Starting from an average diameter of animal cells of between 5 and 15 microns, the diameter of the 2D-MS2 will preferably be between 100 and 300 microns, for example 150 μm. The size of the cell sheets according to the invention will advantageously be between 100 and 300 μm in diameter, and the size of the organoids or spheroids of the order of 50 to 500 μm in diameter. The sheets can consist of one or more cell types.

In a fourth embodiment of the invention, the anchoring-dependent cells, which have been allowed to grow on the 2D-MS2 beyond the confluence stage, form cellular aggregates containing or not containing the starting 2D-MS2 having served in their training. Such aggregates formed naturally without the use of cryosensitivity, are distinguished from spheroids and organoids by their size heterogeneity, as illustrated in Example 5. It should be noted that in this embodiment of the invention; the cryosensitivity of microcarriers is not necessarily used. The cryosensitivity of microsupports can nevertheless be used to unhook all the aggregates of microsupports. It is also important to note that, contrary to what one could imagine, the use of microsupport with three-dimensional geometry does not allow cellular detachment leading to the formation of aggregates, and even less, of spheroids and organoids . In a preferred embodiment of the invention, the microcarriers used in the processes for obtaining cellular aggregates are made from biocompatible and / or bioresorbable materials, of biomedical grade preferably recognized by the FDA, in order to allow implantation in the living organism of aggregates still containing the microcarrier. FIG. 2 represents in schematic form the different methods of cell culture on 2D-MS2 and the products obtained, in accordance with the present invention.

The methods described above can be used to produce individualized cells, cell sheets, spheroids, organoids, or cell aggregates from any type of anchor-dependent cells. They can in particular be fibroblasts, hepatocytes, keratinocytes, pancreatic cells, hematopoietic stem cells or nerve cells. In many cases, the cells thus cultivated will be genetically modified, for example to give them a particular phenotype such as resistance to a chemical agent, the capacity to secrete potentially active substances or the capacity to produce viruses or antibodies.

Of course, the cell aggregates capable of being obtained by any of the methods described in this application, also fall within the scope of the invention as claimed.

The anchor-dependent cells produced according to a method of the invention can be used for the in vitro production of substances of biological, cosmetic, prophylactic or therapeutic interest which are difficult to isolate from organs or physiological fluids. The biological and physiological integrity of the cells produced according to a process of the invention, as well as their high density, makes it possible to produce in large quantities molecules with high added value such as glycoproteins sensitive to the trypsinization of the cells. Examples of substances produced by such cells include antibodies, membrane antigens, membrane receptors, as well as adhesion proteins.

Likewise, the spheroids, organoids or cellular aggregates according to the invention can be used for the in vitro production of substances of biological, cosmetic, prophylactic or therapeutic interest difficult to isolate from organs or physiological fluids. The advantage of these multicellular structures over isolated cells is that the physiological functions of the cells within these structures are improved, or even restored. Furthermore, the spheroids, organoids or cellular aggregates according to the invention can be implanted in the organism of a host, human or animal, and thus serve for the in vivo production of substances of biological, cosmetic, prophylactic or therapeutic interest. . Preferably, cells of a given type will be implanted at the level of the organ or tissue corresponding to the type of cells previously cultivated.

When sheets, spheroids, organoids or cellular aggregates according to the invention are implanted in the organism of a host, human or animal, for the in vivo production of substances of prophylactic or therapeutic interest, these substances can act locally or by systemic diffusion. For example, growth factors and extracellular matrix proteins secreted by heterologous fibroblasts at the level of a skin lesion can stimulate the growth of host keratinocytes and thus promote healing. Furthermore, substances such as erythropoietin or coagulation factors such as factor VIII or factor IX can be synthesized in the skin, in a muscle, or in an organ such as liver or pancreas, and diffuse in the body to exert their action.

In another aspect of the invention, the sheets, spheroids, organoids or cell aggregates are used in the composition of artificial organs or tissues which will then be implanted in a host, human or animal. By "artificial organ" is meant here a relatively ordered set of cells reproducing structures and especially functions of a given organ. They may, for example, be spheroids formed of pancreatic cells reproducing structures close to the islets of Langerhans. They can also be spheroids, organoids or aggregates of liver cells reproducing structures of liver nodules. These artificial organs may contain genetically modified cells. They are then implanted in the corresponding organ, for restoration purposes when this organ has been injured, or for correction purposes when this organ is deficient. Thus, in cases of diabetes, islets of artificial Langerhans can be implanted in the pancreas of the patients, in order to correct their insulin secretion. The implantation of artificial micro-organs, comprising genetically modified cells, can also aim to correct a deficiency which is not linked to this organ. For example, hepatocyte spheroids producing a high level of coagulation factor (Factor VIII or Factor IX) can be grafted into the liver of hemophiliac patients with a deficiency of said factor.

An “artificial tissue” is defined here as a composition comprising sheets, spheroids, organoids or cellular aggregates corresponding to a given type of tissue, such as the skin or the muscle, for example. As with artificial organs, artificial tissues are intended to be implanted in the host, human or animal, at the level of the corresponding tissue, for the purpose of restoring damaged tissue, or for the purpose of grafting cells producing a prophylactic or potentially therapeutic substance for the host. Particular examples of artificial tissues according to the invention are presented in Examples 3 and 4. In another application, the cells constituting the artificial tissue (keratinocytes and / or fibroblasts, for example), can be genetically modified to produce a substance d interest to the host. Mention will in particular be made of erythropoietin, factor VIII, factor IX.

Such artificial organs or tissues are also part of the invention. In a particular embodiment of the invention, micro-sheets produced from allogenic and / or autogenic human keratinocytes on 2D-MS2 are used as such or incorporated into a formulation for the treatment of chronic wounds (ulcers, bedsores) or acute (burns) of the skin. It should be noted at this stage that the 2D-

MS2 can be coated or covalently bonded (grafted) with collagen, fibronectin or poly-L-Lysine to improve adhesion of keratinocytes. In the case of chronic or acute wounds, the application to the wound of bioactive dressings made up of allogenic cells such as fibroblasts or keratinocytes allows better healing and prepares it for a possible graft of autologous cells (patient's cells) while limiting dehydration and infections when the preparation is applied under a waterproof bandage (silicone film for example). Most of the skin substitutes developed so far consist of three-dimensional matrix of collagen, nylon, vicryl, hyaluronic acid on which fibroblasts, keratinocytes or both are cultivated (Yannas et al., 1982; Damour et al., 1994; Naughton, 1997; US patent 5,166,187; US patent 5,266,480). The most widely used skin substitute however remains the simple sheet of autologous keratinocytes prepared from a biopsy taken from the patient (O'Connor et al., 1981; Gallico et al., 1984; Cuono et al., 1986). Only Boehringer Mannheim has developed a culture system for epithelial cells on 3D microcarriers for the purpose of transplants (WO 96/12510). This system, which most closely resembles that described here, however presents several imperfections with respect to the present invention. On the one hand, the microsupports used are three-dimensional and therefore do not lodge perfectly on the wounds, which implies a transfer of very random growth factors and cells. On the other hand, since the microcarriers are not bioresorbable, they will remain on the wounds and require removal before the wound cells cover them and are thus irreparably damaged. "Incorporated" into the body. In a particularly preferred embodiment of our invention, allogenic keratinocytes are cultured on 2D-MS2 until confluence. Once the interkeratinocyte junctions have been formed, the micro-sheets are unhooked by thermal contrast and then incorporated into a gelled formulation.

(whether or not containing antibiotics), then applied to the wound. A bandage (silicone for example) is made to limit dehydration and infections. This method is particularly suitable for the treatment of burns but also of ulcers and bedsores, as described in example 4. In the case of deep burns where few keratinocytes of the patient are likely to reform an epidermis, autologous cells (of the patient) or a mixture of autologous and heterologous cells can be grown on the 2D-MS2 microcarriers. In another preferred embodiment of the invention, these are fibroblast micro-sheets which are generated from 2D-MS2 microcarriers, as illustrated in example 3. These are more particularly used for the treatment of ulcers and bedsores but also for the recovery of burns awaiting a transplant of autologous keratinocytes.

Important applications of cells, sheets, spheroids, organoids, or cell aggregates according to the invention are the fields of pharmacology and toxicology. Organoids are of particular interest for these applications. Indeed, the particular membrane integrity properties of cells, and the organized structure of organoids, make them interesting models for carrying out pharmacological and toxicological tests. In particular, they offer tools of choice for screening pharmacologically active molecules, particularly when these act on organized structures such as membrane receptors, or intracellular junctions. Example 5b shows the production of organoids consisting of a heart of fibroblasts surrounded by an external gangue of keratinocytes. In a preferred embodiment of the invention, normal human melanocytes are also incorporated into the external gangue of the organoid in a proportion of 1-5 melanocytes per 10 keratinocytes. We then obtain pigmented organoids composed of the three main cell types that make up the skin. Immune cells from

Langerhans, as well as dermal endothelial cells, can also be incorporated to build an even more complete system. Such organoids in a way reproduce human skin, and therefore constitute particularly interesting models for carrying out pharmacological and toxicological tests. This approach can of course be used to produce models for other tissues and / or organs.

The cells, sheets, spheroids, organoids or cell aggregates according to the invention can also be used for the development of diagnostic tests and the manufacture of vaccines. In particular, the individualized cells obtained by a method as described above and illustrated in Example 1, have intact membrane proteins. They can therefore be advantageously used for the extraction and purification in particular of receptors and other membrane antigens useful for the development of diagnostic tests, and for the manufacture of immunogenic compositions or of vaccines.

Another possible field of application of a CAD culture method according to the invention is the production of viral vectors for gene therapy. A first application consists, for example, in cultivating transcomplementing cells for defective adenoviruses according to a continuous or non-continuous process, as described above. When the cells have reached the optimal confluence for the infection (which may depend on the cells concerned), they are put in the presence of the virus to be amplified, at a multiplicity of infection such that a large proportion of the cells are infected. The high cell density obtained by the methods according to the invention makes it possible a priori to infect a large proportion of the cells in using a relatively small viral inoculum. For the same reason, the virus recovered in the supernatant, after the lysis of the cells at the end of the viral cycle, will a priori be more concentrated when it is produced in other systems.

Another possible application of the methods of the invention in gene therapy is the production of spheroids, organoids or aggregates of cells producing retroviral or lentiviral vectors. These vector-producing spheroids, organoids or aggregates are then implanted in the human or animal host, at the site to be treated. They may, for example, be cells producing vectors encoding a neuroprotective factor, to be implanted in the brains of patients suffering from neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. It can also be cells producing vectors coding for a gene having a potential anti-tumor activity (“suicide” gene such as HSV thymidine kinase, cytokine gene, p53 gene, etc.). In this case, the spheroids, organoids or aggregates producing said vectors are implanted near the tumor, possibly after its total or partial resection. In this application, the spheroids, organoids or aggregates according to the invention have the advantage of being more easily implantable than isolated cells.

The examples below illustrate the implementation and the interest of the present invention, without however limiting its scope. Legend of figures:

FIG. 1 represents in schematic form the processes for obtaining cell sheets and spheroids according to the invention.

FIG. 2 represents in schematic form the different methods of cell culture on 2D-MS2 and the products obtained, in accordance with the invention described above.

Figure 3 is a photograph of a microcarrier covered by fibroblasts.

FIG. 4 is a photograph of a microcarrier coated with human γ-irradiated fibroblasts and seeded with keratinocytes.

FIG. 5 is a photograph of cell aggregate obtained by culture on 2D-MS2 of fibroblasts, beyond the stage of confluence, and surrounding the microcarrier.

FIG. 6 is a photograph of a cell aggregate obtained by culture on 2D-MS2 of fibroblasts, beyond the stage of confluence, dissociated from the microcarrier.

EXAMPLE 1 Culture of Anchor-Dependent Cells on Cryosensitive Microsupport with Two-Dimensional Geometry (2D-MS2) for the Production of Individualized Intact Cells

0.55 g of 2D-MS2 (760 cm ² / g) corresponding to a total surface of 418 cm ² are mixed with 8.36.10 ⁶ human fibroblasts (20,000 cells / cm ² ) in a final volume of 50 ml of medium of culture DMEM / F12 (1: 1) supplemented with 10% of newborn serum (NCS) and the whole placed in a culture flask with pendulum agitation for culture on microsupport. The stirring speed is set at 25 rpm for 16-18 h so that the cells can adhere to the microcarriers, the culture flask being gassed continuously with a mixture of air / CO2 (95% -5% v / v) . After this period, the volume of the culture is brought to 100 ml and the stirring speed brought to 40 rpm. The culture is maintained under these conditions for one week with daily change of medium. At this time, the cells (which have not yet reached confluence) are detached by thermal contrast by lowering the temperature of the culture medium to 4-10 ° C. After 15 minutes under these conditions (the agitation is not interrupted), all of the detached cells are separated by sedimentation of the microcarriers. The percentage of viability determined by trypan blue staining is 95%. This technique can be extrapolated to culture volumes more related to an industrial scale. The cells thus obtained, the membrane proteins of which are intact, can be used for the extraction and purification, in particular of membrane receptors which can be used for the development of diagnostic tests, for the manufacture of vaccines and in fundamental research. Example 2 Serial Culture of Anchor-Dependent Cells on Cryosensitive Microcarrier

The serial culture of anchor-dependent cells in bioreactors on cryosensitive microcarriers is made possible without the intervention of a proteolytic enzyme (trypsin, dispase, thermolysin), by alternating cell growth cycles, partial cell harvest cycles (by thermal contrast) and again growth cycles of the non-harvested cells which are used to re-inoculate the microcarriers.

L929 mouse fibroblastic cells are inoculated into a 3 I bioreactor (11 of medium) at a rate of 20,000 cells / cm ² of partially (95-98%) cryosensitive two-dimensional microsupport, in 10% NCS DMEM / F12 medium. The medium is renewed at the rate of 2 l / d by infusion. The growth of these cells on the microcarriers is monitored daily by microscopic observation and measurement by Aperture Impedance Puise Spectroscopy (Degouys et al., 1993). Before confluence, the cells are detached by thermal contrast by lowering the temperature of the culture medium to 4 ° C for 15 minutes by injecting ice water into the jacket of the bioreactor. After decanting the microsupports, the individual cells in suspension are collected and the microsupports carefully washed with cold culture medium.

The cells that remain attached to the non-cryosensitive regions of the 2D-MS2 constitute the inoculum intended for the next growth cycle. This cycle is started by the addition of culture medium at 37 ° C. A growth cycle is then started again. Example 3: Use of two-dimensional cryosensitive microcarriers

(2D-MS2) for the production of micro-sheets of human fibroblasts for the therapy of chronic (ulcers, bedsores) and acute (burns) wounds of the skin (see photo of Figure 3)

Allogenic human fibroblasts are inoculated on 2D-MS2 cryosensitive microcarriers in pendulum shaking culture flasks (Techne or Wheaton brand) at a rate of 10-20 cells per microsupport (5.10 ⁴ -10 ⁵ cells / ml) in 75 ml of DMEM / F12 (1: 1) containing 10% Newborn Calf Serum "NCS" (Newborn Calf Serum). The cells and the microcarriers are agitated at 25 rpm overnight, then the following day, the volume of medium is doubled and the agitation brought to 40 rpm. The culture medium is changed every day. The fibroblast culture is stopped when they have a density of microcarrier coverage of 100% (see photo in Figure 3). By lowering the temperature of the culture medium, fibroblast micro-sheets are detached from the microcarriers, rinsed and then applied as such to the wounds or included in a gelled formulation (methylcellulose, agarose, etc.). The micro-sheets can also be frozen and stored at - 80 ° C or - 196 ° C in a solution preferably containing as cryopreservative

DMSO, glycerol or trehalose.

The harvested micro-sheets are preferably rinsed in medium without serum before being applied directly to the wound as such or included in a formulation in gelled form (methyl cellulose, agarose, etc.). A bandage is then affixed to the wound to prevent dehydration and infections. In this way, once the micro-sheets are applied to the wound, the growth factors and proteins of the extracellular matrix produced by these cells can diffuse more easily and the cells migrate from the micro-sheet to the wound in a few hours. A once adhered to the wound, they continue to synthesize and diffuse their growth factors and proteins from the extracellular matrix. This embodiment of the invention is particularly suitable for the treatment of chronic wounds (ulcers, bedsores) but can also be used for the treatment of burns.

Example 4: Use of two-dimensional cryosensitive microcarriers

(2D-MS2) for the production of micro-sheets of human keratinocytes for the therapy of chronic wounds

(ulcers, bedsores) and acute (burns) of the skin (see photo in Figure 4)

In a preferred embodiment of the invention, human keratinocytes are seeded on a sublayer of irradiated γ human fibroblasts (or mitomycines) in order to stop their growth without altering their membrane properties or metabolism, cultured on 2D microcarriers -MS2, at a rate of 10-20 KHN (Normal Human Keratinocytes) per microcarrier in a volume of culture medium as small as possible. For example, 2.10 ⁶ KHN are seeded on 2.10 ⁵ 2D-MS2 (0.6 cm ² ) in a volume of 10 ml of DMEM / F12 medium, 10% NCS, containing hydrocortisone 0.4 μg / ml, EGF 10 ng / ml, transferrin 5 μg / ml, triiodothyronine (2 nM), adenine 0.18 mM, cholera toxin 0.1 μg / ml and insulin 5 μg / ml in a bacteriological petri dish (not treated for cell culture) or in a 50 ml conical tube. The petri dish or the tube is placed in a CO ₂ incubator at 37 ° C. and are shaken every Vz hours for a few seconds. At the end of 4 p.m. to 6 p.m., we can consider that all the NHKs which have not joined will no longer join. The microcarriers are then transferred to a spinner (25-50 ml) or a pendulum shaking flask (125 ml) and minimal agitation of the microcarriers is ensured so that they do not settle. Conversely, if the agitation is too strong, the cells fall off. The culture medium volume is brought to 25-50 ml. The KHNs are allowed to grow until they cover 100% of the surface of the microcarriers and in particular until the interkeratinocyte junctions are strong (3-4 days after the confluence in a hypercalcic medium). By lowering the temperature of the culture medium, micro-sheets of keratinocytes are detached from the microcarriers, rinsed and then applied as such to the wounds or included in a gelled formulation (methylcellulose, agarose, etc.). The micro-sheets can also be frozen and stored at - 80 ° C or - 196 ° C in a solution preferably containing as cryopreservative of DMSO

(10%), glycerol (10%) or trehalose (0.2-0.4M).

EXAMPLE 5 Use of a Cryosensitive Microsupport with Two-Dimensional Geometry for Culturing Anchor-Dependent Cells in the Form of Spheroids

This example illustrates how the use of two-dimensional cryosensitive microcarriers can be used to form spheroids (or organoids).

5a. Human fibroblast spheroids.

Normal human fibroblasts cultured in culture flasks in DMEM / F12 medium (1: 1), 10% NCS are individualized by trypsinization

(trypsin / EDTA 0.05%: 0.02% pH 7.4) then inoculated at the rate of 10 ⁴ - 10 ⁵ (preferably 4.10 ⁴ ) cells / cm ² of microsupport in a culture flask with pendulum shaking ( Techne, Cambridge UK) in DMEM / F12 medium (1: 1), 10% NCS (75 ml total). The Techne is stirred (25 rpm) at 37 ° C and gassed by an air / CO ^ (95% / 5%) flow for

24 hrs for cell anchoring. The next day, the stirring is brought to 40 rpm and culture medium is added (150 ml final). At confluence (approximately 1 week after seeding), the cells are detached by thermal contrast by rapidly cooling the culture medium to a temperature of 4-10 ° C for about 15 minutes with controlled stirring. The agitation is then stopped and the microcarriers are left to settle for a few minutes. The result obtained consists of micro-sheets (single or multi-layer) or cellular flakes the size of the microcarriers used (in this case 150 μm in diameter). These microsheets are then removed, then returned to agitation without microsupport until homodisperse spheroids consisting of fibroblasts are obtained.

5b. Organoids composed of fibroblasts and human keratinocytes

Organoids based on fibroblasts and human keratinocytes can be produced according to a slightly more complex procedure. First, fibroblast spheroids are made as above. When these have formed, the culture medium is replaced by another having the following composition: DMEM / F12 (3: 1) containing 10% of NCS, hydrocortisone 0.4 μg / ml, EGF 10 ng / ml, transferrin 5μg / ml, triiodothyronine (2 nM), adenine 0.18 mM, cholera toxin 0.1 μg / ml and insulin 5μg / ml. In a preferred embodiment of the invention, the fibroblasts used during the inoculation of the microcarriers are mitomycin fibroblasts or which have undergone γ irradiation, these then no longer being able to multiply. Human keratinocytes are then seeded (50-100,000 KHN / ml) on these fibroblastic spheroids. The culture is thus continued for another one or two weeks, the keratinocytes cover the surface of the spheroids, then begin to form several layers, differentiating themselves. The organoid thus obtained consists of a spheroid whose heart of fibroblasts is surrounded by an external gangue of keratinocytes. Each of these organoids in a way reproduces human skin and the thousands of organoids obtained can constitute interesting models for carrying out pharmacological and toxicological tests for pharmaceutical companies, cosmetics and academic laboratories. Optionally, normal human melanocytes are also incorporated into the external gangue of the organoid at a rate of 1-5 melanocytes per 10 keratinocytes. BFGF is then incorporated into the culture medium at a rate of 1 ng / ml. We then obtain pigmented organoids composed of the three main cell types that make up the skin. Langerhans immune cells, as well as dermal endothelial cells can also be incorporated to build an even more complete system.

5c. Pig or rat Langerhans islet cell spheroids

Pancreatic islet cells from pig or rat Langerhans can be cultured as in Example 3a and form functional spheroids capable of synthesizing insulin. These spheroids can be used encapsulated in a system impermeable to the patient's antibodies, for therapeutic purposes (grafts) in insulin-dependent diabetics.

Example 6: Use of two-dimensional microcarriers to carry out the culture of anchor-dependent cells in the form of aggregates (three-dimensional structure and heterodisperse size distribution) (see photos of FIGS. 5 and 6)

0.55 g of two-dimensional microcarriers (760 cm ² / g), i.e. 418 cm ² are inoculated in DMEM / F12 medium (1: 1) 50 ml with 8.36.10 ^e fibroblasts (20,000 c / cm ² ) in a culture flask for culture on microsupport (Wheaton or Techne brand).

The stirring speed is set at 18 rpm for 16-18 h so that the cells can adhere to the microcarriers and the culture flask is continuously gassed with a mixture of air / CO ₂ (95/5%). The next day, 50 ml of additional medium are added and the stirring speed is brought to 40 rpm.

The culture is maintained for 15 days at 37 ° C. under a 5% CO ₂ atmosphere, the culture medium being changed every day.

After 15 days, the aggregates are harvested by sieving on a sieve of 125 μm in diameter. This collects cellular aggregates formed around a mold which is constituted by the microcarrier (FIG. 5), and aggregates which have dissociated from the microcarrier (FIG. 6).

References cited:

Carvalhal, AN., Moreira, J.L., Miranda, P.M. and Carrondo, M.J.T. (1997) Hydrodynamic control of aggregate size in suspended cultures of different animal cells. Animal Cell Technology (Kluwer Academy Publishers), 779-784.

Cuono, C, Langdon, R. and McGuire, J. (1986). Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancefv. 1123-1124.

Damour, O., Gueugniaud, P.Y., Berthin-Maghit, M. Rousselle, P., Berthod, F., Sahuc, F. and Collombel, C. (1994). A dermal substrate made of collagen-GAG-Chitosan for deep burn coverage: first clinical uses.

C // t7 / ^' ca / Materials. 15: 273-276.

Degouys, V., Cerckel, I., Garcia, A., Harfield, J., Dubois, D., Fabry, L. and Miller, AO. (1993) Dielectric spectroscopy of mammalian cells. Simultaneous in situ evaluation by aperture impedance draws spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on cytodex 3 Cytotechnology, 13. 195-202.

Gallico, G. G., O'Connor, Ν.E., Compton, C.C., Kehinde, O. and Green, O. (1984). Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med. 31 1: 448-451.

Ijima, H., Matsushita, T., Nakazawa, N., Koyama, S., Gion, T., Shirabe, K., Shimada, M., Takenaka, K., Sugimachi, K. and Funatsu, K. ( 1997) Spheroid formation of primary dog hepatocytes using polyurethane foam and its application to hybrid artificial liver. Animal Cell Technology (Kluwer Academy Publishers), 577-583. Koide, N., Shinji, T., Tanabe, T., Asano, K., Kawaguchi, M., Sakaguchi, K., Koide, Y., Mori, M., Tsuji, T. (1989) Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem. Biophys. Res. Comm. 161, 385-391.

Matsushita, T., Ijima, H. and Funatsu, K. (1992) Development of a hybrid artificial liver utilizing three dimensional culture of adult hepatocytes.

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Matsushita, T., Ijima, H., Koide, N. and Funatsu, K. (1991) High albumin production by multicellular spheroid of adult rat hepatocytes formed in the pores of polyurethane foam. Appl. Microbiol. Biotechnol. 36, 324-326.

Naughton, G.K. (1997). Skin and epithelia. Principles of tissue engineering. Robert Lanza editors. Academy press California USA. pp 769-782.

O'Connor, N., Mulliken, J.B., Banks-Schlegel, S., Kehinde, O. and Green, H. (1981). Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet i: 75-78.

Takezawa, T., Mori, Y., Yonaha, T. and Yoshizato, K. (1993) Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts. Exp. Cell. Res. 208, 430-441.

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Takezawa, T., Yamasaki, M., Mori, Y., Yonaha, T. and Yoshizato, K. (1992). Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes. J. Cell Sci. 101. 495-501. Tong, JZ, Bernard, O. and Alvarez, F. (1990) Long-term culture of rat liver cell spheroids in hormonally defined media. Exp. Cell. Res. 189, 87-92.

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Claims

1. Method for obtaining anchor-dependent cells (CAD), comprising the following steps: a) inoculation of cryosensitive microsupports with two-dimensional geometry (2D-MS) by CAD, b) culture at a temperature above the critical lower temperature (LCST) of the polymer responsible for the microsupport's cryosensitivity, c) detaching the cells from the microcarriers, by lowering the temperature of the culture to a temperature substantially lower than said LCST, d) if necessary, reproducing steps b) and c ) until the desired amount of cells is obtained.

2. Method for obtaining CAD according to claim 1, characterized in that the microcarriers seeded in step a) consist of a polymer film onto which are molecules covalently grafted the molecules ensuring the cryosensitivity of said microcarriers.

3. Method for obtaining CAD according to claims 1 and 2, characterized in that the entire surface of the 2D-MS2 is cryosensitive and all the cells are detached from the microcarriers in step c).

4. Method for obtaining CAD according to claims 1 and 2, characterized in that the microcarriers are partially cryosensitive, and that step d) is carried out by resuspension at 2D-MS culture temperature after detachment of the cells according to step c).

5. Method according to claim 4, characterized in that 2 to 10% of the surface of 2D-MS are not cryosensitive.

6. Method according to claim 4, characterized in that the thickness of the polymer deposit is such that 2 to 10% of the cells do not detach from the microcarrier during step c).

7. A process for obtaining CAD according to claims 1 and 2, characterized in that the microcarriers are previously covered with a sublayer of anchoring-dependent cells of a different nature from that of the cells innoculated in step a).

8. A process for obtaining CAD according to any one of claims 1 to 3 and 7, in which the stage b) of culture is continued until the stage of confluence, and the CAD are obtained after stage c ) in the form of cellular sheets.

9. The method of claim 8, wherein the size of the sheets obtained after step c) is controlled.

10. Process for obtaining cellular aggregates comprising the following steps: a) inoculation of 2D-MS2, if appropriate biocompatible and / or bioresorbable with CAD, b) cultivation at a temperature above the critical lower temperature (LCST) of the polymer responsible for the cryosensitivity of the microcarrier, beyond the confluence stage, c) if necessary, detachment of the cells from the microcarriers, by lowering the temperature of the culture to a temperature substantially lower than said LCST.

11. A process for obtaining spheroids or organoids, comprising the following steps: a) recovery of cell sheets obtained by a process of claim 8 or 9, b) resuspension of these sheets in culture medium at 37 ° C, c) elimination of 2D-MS from the culture medium, this step being feasible before or after step b).

12. The method of claim 11, wherein the cell sheets recovered in a) are homodispersed in size, and the size of the spheroids or organoids obtained is controlled.

13. A process for obtaining complex organoids comprising the following steps: a) obtaining spheroids or organoids by a process according to claim 11 or 12, b) inoculating these spheroids or organoids with other CAD, of the same cell type , or of a different cell type, c) continuing the culture until the surface of the spheroids or organoids of step a) is covered by the cells seeded in b), d) where appropriate, the steps b) and c) can be repeated on the complex organoids obtained in c) by seeding the latter with cells of a type different from that of b).

14. Process for obtaining complex organoids according to claim 13, in which the spheroids of step a) consist of fibroblasts which have undergone a treatment to render them incapable of multiplication, and the CAD of step b) are mainly keratinocytes.

15. Suspension of cell sheets capable of being obtained by a process according to claim 8 or 9, characterized by the uniformity of size of the sheets, the membrane integrity of the cells making up these sheets and their viability.

16. Suspension of cell sheets according to claim 15, such that the size of the sheets is between 100 and 300 μm in diameter.

17. Spheroids or organoids of anchor-dependent cells, capable of being obtained by a process according to claim 11, 12 or 13.

18. Suspension of spheroids or organoids of anchoring-dependent cells according to claim 17, such that the size of the spheroids or organoids is between 50 and 500 μm in diameter.

19. Organoids according to any one of claims 17 and 18, characterized in that they comprise cells of fibroblast and keratinocyte type.

20. Cellular aggregates capable of being obtained by a method according to claim 10.

21. Cell sheets, spheroids, organoids or aggregates according to any one of claims 15 to 20, consisting at least in part of genetically modified cells.

22. Sheets, spheroids, organoids or cell aggregates according to any one of claims 15 to 21, consisting of fibroblasts, keratinocytes, melanocytes, hepatocytes, myoblasts, pancreatic, nerve cells or a mixture of those -this.

23. Anchor-dependent cells, capable of being obtained by a process according to any one of claims 1 to 6, or cell sheets, spheroids, organoids or aggregates according to any one of claims 15 to 22, for the production of substances of biological, cosmetic, prophylactic or therapeutic interest.

24. Anchor-dependent cells, cell sheets, spheroids, organoids or aggregates according to any one of claims 21 to 23, for the production of viral vectors for gene therapy.

25. Artificial organ or tissue comprising sheets, spheroids, organoids or cell aggregates according to any one of claims 15 to 22, and pharmaceutically acceptable excipients.

26. An artificial organ or tissue according to claim 25, for the secretion of substances for therapeutic or prophylactic purposes for the host.

27. Use of an artificial organ or tissue according to claim 25 or 26; in the manufacture of a therapeutic composition for repairing the corresponding organ or tissue in a human or animal host.

28. Use of sheets, spheroids, aggregates or cellular organoids according to any one of claims 15 to 22, or artificial tissue according to one of claims 25 or 26, for the preparation of a therapeutic composition intended for the treatment of burns and chronic wounds.

29. Use of sheets, spheroids, organoids or cell aggregates according to any one of claims 15 to 22, for carrying out pharmacological or toxicological tests in vitro.

30. Use of CAD, sheets, spheroids, organoids or cell aggregates according to any one of claims 15 to 22, for the manufacture of reagents for diagnostic tests or the manufacture of immunogenic compositions.

31. Sheets, spheroids, organoids or cellular aggregates according to any one of claims 15 to 22, comprising pancreatic cells if necessary recombined, for their implantation in the pancreas, for the regulated secretion of insulin.