EP2250253A1 - System and method for the clonal culture of epithelial cells and applications thereof - Google Patents

Abstract

The invention relates to means and methods for evaluating and using the specific properties of a particular epithelial cell present in a biological sample. Accordingly, the invention relates to a system for the culture of epithelial cells, in which at least one clonal culture is sown with a single epithelial cell directly extracted from a biological sample of epithelial tissue. The invention also relates to a method for the culture of epithelial cells, that particularly comprises the production of clonal cultures, each being sown with a distinct and unique epithelial cell directly extracted from a biological sample of epithelial tissue, the evaluation of the cellular growth in the clonal cultures, and advantageously the analysis of the capacity of the cellular material from the clonal cultures to reconstruct a three-dimensional epithelium representative of native tissue. The invention is adapted for the parallel implementation of a very large number of clonal cultures, in particular for making large-scale tests.

Description

 SYSTEM AND METHOD FOR CLONAL CULTURE OF EPITHELIAL CELLS AND THEIR APPLICATIONS

The present invention relates to the field of cell biology and tissue engineering.

More specifically, the invention provides means and methods for evaluating and exploiting the specific properties of a particular epithelial cell present in a biological sample.

Thus, the present invention relates to an epithelial cell culture system, wherein at least one clonal culture is seeded with one and only one epithelial cell directly extracted from a biological sample of epithelial tissue.

The invention further relates to a method of culturing epithelial cells, comprising at least the steps of: a) extracting one or more epithelial cells directly from a biological sample of epithelial tissue; b) optionally, selecting at least one population and / or subpopulation of epithelial cells from the cells extracted in step a); c) performing a clonal culture seeded with a distinct and unique epithelial cell directly from step a) or b); and d) qualitatively and / or quantitatively assessing cell growth in the clonal culture of step c).

In addition, the invention is directed to the applications of such a system or method. In vitro systems and methods for epithelial tissues such as the epidermis have applications in areas as diverse as medical research and clinical development, tissue engineering and toxicology.

The different pluristratified epithelial tissues (in particular the epidermis, the cornea, the mucous tissues, etc.) share a certain number of common characteristics which impose constraints for the design of large scale in vitro test architectures. These general characteristics are well exemplified in the case of the epidermis.

The epidermis constitutes the most superficial structure of the skin and in particular ensures the barrier function. Mostly consisting of keratinocytes, it is renewed every 28 days on average. This tissue comprises 4 layers that correspond to the 4 stages of the differentiation program that the keratinocytes undergo during their migration from the basal layer, the deepest, to the stratum corneum, the most overperfective. The continuous physiological process of renewal of the different layers of keratinocytes is called keratinopoiesis.

The basal layer of the epidermis, which has only one cell base, is the germinal compartment. It is at the level of this layer that the proliferation of keratinocytes takes place. Among the basal keratinocytes, there is a small proportion of cells called stem cells, which are believed to be responsible for long-term epidermal renewal. The immediate offspring of stem cells is called the progenitor population. These ensure the rapid renewal of the epidermis in the short term. The notion of stem cell, within the human inter-follicular epidermis, therefore defines the compartment located furthest upstream of the hierarchy of keratinopoiesis. These cells are characterized in particular by a high self-renewal capacity, which the progenitor cells do not possess, and a fortiori the keratinocytes involved in the differentiation. In addition, an important property of stem cells is to sustainably conserve the potential for regeneration and reconstruction of the epidermal tissue.

Like other pluristratified epithelial tissues, the epidermis thus consists of a heterogeneous set of cells with variable degrees of differentiation (or immaturity). It is generally accepted that basal keratinocytes represent approximately 10% of the whole of the keratinocytes of the epidermis, and the epidermal stem cell compartment only of the order of 0.1%.

In view of the quantitative requirements necessary for the implementation of current functional evaluation and screening methods, the cellular material routinely collected from cutaneous biopsies, namely all the keratinocytes obtained after dissociation of a sample of epidermis, allows the constitution of cell banks of sufficient size for use on an industrial scale. However, the material used for the constitution of this type of library corresponds to a heterogeneous set of cells, comprising basal keratinocytes having different growth capacities, and supra-basal keratinocytes being differentiated and no longer having growth capacity.

As regards the keratinocyte libraries, for example intended for the industrial production of reconstructed epidermis kits, a conventional method consists in freezing the cells at the end of a single multiplication stage in culture, so as to constitute a stock multiple equivalent ampoules, which are kept in liquid nitrogen. As needed, the ampoules of cells are thawed and placed in culture to perform a second stage of multiplication. At the end of the two successive multiplication steps, the keratinocytes have generally performed in the order of 10 population doublings. This type of approach has been used to analyze the heterogeneity of the growth potential of human keratinocytes (Barrandon and Green, 1987). The authors first made primary cultures derived from the epidermis, which they frozen in liquid nitrogen. Subconfluent side crops were prepared from the frozen primary cultures. The clones obtained after cloning (third stage of culture or "third passage") were classified into three categories: holoclones (fast growing), paraclones (limited growth) and meroclones (intermediate population). The keratinocytes conventionally obtained after two successive multiplication steps can be used for the production of reconstructed tissue models. Applying these systems as such to a rare cellular material, such as stem cells, is however impossible on an industrial scale where extensive testing campaigns must be conducted.

In sum, several types of models compatible with large-scale in vitro test campaigns are currently available. The biological material used in these tests may be: 1) immortalized cell lines; 2) normal cell banks extracted from tissue biopsies and amplified in culture; 3) reconstructed three-dimensional tissues. However, for the large scale implementation of such models, it is necessary to have large quantities of cellular material. For example, in the case of tests performed on normal cells, cell populations amplified in vitro are used, which modifies certain properties as a function of the culture parameters applied. In addition, if one is interested in subpopulations of rare cells, such as progenitor cells or epidermal stem cells, these cells are obtained in insufficient quantities from tissue biopsies.

Gangatirkar et al. (2007) describe a method for reconstructing an epidermis from total keratinocytes or from subpopulations sorted on the basis of distinct phenotypes. The two proposed options consist, on the one hand, in using the cells directly after extraction from the tissue and cell sorting, and, on the other hand, in using the cellular material after an expansion phase in culture. However, the quantitative requirements for cellular hardware to implement the described method remain unsuitable for carrying out large scale parallel testing campaigns. In addition, the cellular material used corresponds to a complex mixture of different cells whose ability to reconstruct an epidermis is used in a global manner. The heterogeneity of the cells composing the pleniscated epithelial tissues, and in particular that of the keratinocytes of the epidermis, is therefore a limiting factor in the development of in vitro test strategies.

Indeed, since cells have certain characteristics of their own, they are likely to respond differently to a stimulus or stress. It is the same for pathological epithelial tissues. For example, carcinomas are very heterogeneous tumors, in which a small proportion of tumor stem cells represent a key target for treatment. In addition, when cells having distinct characteristics are mixed in culture, the specific behavior of some of them can be modified or ignored within the mixture. An "average" result is thus observed on all the cells cultured. Thus, the structural and functional properties of a reconstructed epidermis from a heterogeneous global cell population (see for example Larderet et al., 2006) are the result of all the properties of the cells involved. An epidermis thus reconstructed can not in any case reflect the specific properties of a single cell. In addition, the culture conditions applied can more or less severely modify the intrinsic characteristics of the cells. It is well known that placing cells derived from an epithelial tissue in an artificial culture environment leads to modifying their native characteristics. Consequently, epithelial cells used after one or more culture steps represent cell material that is no longer comparable to cells directly derived from a tissue sample. These modifications concern in particular the specific phenotype of the cells studied. For example, it has been shown that culturing human keratinocytes freshly isolated from an epidermis disrupts the expression of adhesion molecules and markers used to define a stem cell phenotype. in a variable manner depending on the culture medium used (Lorenz et al., 2008).

Ultimately, conventional solutions to work from heterogeneous complex cell populations are not adequate to meet current medical, clinical and industrial needs.

There is therefore a need for means and methods which make it possible to access the individual properties of the cells derived from the pluristratified epithelial tissues, while being adapted to the implementation of large scale in vitro test campaigns, even in the case of a cell material 100 to 1000 times rarer than the all-round populations that are currently used.

The present invention responds for the first time to this need by proposing means and methods of culture which (i), by their clonal character, allow access to the individual and specific properties of cells directly derived from pluristratified epithelial tissues, (ii) ) preserve the individual potential of said cells, (iii) are, even when implemented on a large scale, low consumption of cellular material, making them suitable for the study and exploitation on an industrial scale of the least represented cells (stem and progenitor cells), and (iv) allow to reach cell growth levels much higher than those obtained with the known tools.

Thus, an object of the present invention is directed to an optimized clonal epithelial cell culture system for evaluating and exploiting the unique properties of a single cell, wherein a culture support comprises at least one clonal culture seeded with a single epithelial cell directly extracted from a biological sample of epithelial tissue. Advantageously, said culture support comprises at least two parallel clonal cultures, each of said cultures being seeded with a distinct and unique epithelial cell directly extracted from said biological sample.

Preferably, such a system is in the form of a biochip. Clonal cultures seeded in parallel are then for example micro-cultures. In practice, the biochip may in particular be made from culture plates comprising multiple distinct wells, for example 6, 24, 96 wells or more. Alternatively, the biochip may have as support a slide, made of glass or any other suitable material, on which multiple micro-surfaces are created for receiving the cells, for example by a surface treatment allowing the cells to adhere and grow. Microchips made on slides can be physically compartmentalised, for example using grids, or chemically, for example following a surface treatment of the slides which prevents the cloned cells from migrating outside their respective micro-culture surfaces.

Another object of the present invention is an optimized clonally cultured epithelial cell culture method for evaluating and exploiting specific properties of a single cell, comprising at least the steps of: a) extracting one or more epithelial cells directly from a biological sample of epithelial tissue; b) optionally, selecting at least one population and / or subpopulation of epithelial cells from the cells extracted in step a); c) performing a clonal culture seeded with a distinct and unique epithelial cell directly from step a) or b); and d) qualitatively and / or quantitatively assessing cell growth in the clonal culture of step c).

Interestingly, the cells used in the method of the present invention may be total populations of cells directly extracted from these tissues, and / or subpopulations thereof, sorted on the basis of precise characters. Thus, the cellular material of step b) may advantageously correspond to one or more subpopulations enriched in progenitors and / or epithelial stem cells.

In step c) (which may be considered as a primary growth step), the cell preparation thus extracted is used to initiate parallel clonal cultures or micro-cultures. It involves inoculating the cells of interest individually under conditions allowing their growth, for example in separate culture wells. In practice, clonal seeding can be carried out in an automated manner by technologies such as, in particular, flow cytometry or microfluidics. Preferably, step c) comprises the production of at least two parallel clonal cultures, each of said cultures being seeded with a distinct and unique epithelial cell directly derived from step a) or b). In particular, during step d), the growth of the cloned cells is analyzed on the basis of one or more quantitative and / or qualitative parameters such as:

the frequency of clones obtained relative to the number of seeded cultures: clone-forming efficiency (CFE); the proliferative potential of the clones: number of cells composing each clone at a given culture time;

the phenotype of the clones: degree of differentiation of the cells composing the clones, expression of molecular markers.

Thus, contrary to all expectations, the inventors have been able to observe that the growth potential of the cells cultured according to the clonal culture method of the present invention is higher than that of cells cultured according to conventional procedures (see Example B below). .

In a particular embodiment, the clonal culture method of the invention comprises at least the steps of: a) extracting one or more epithelial cells as a single cell suspension (s) directly from a biological sample of epithelial tissue; b) selecting at least one population and / or subpopulation of epithelial cells from the cells extracted in step a); c) performing a clonal culture seeded with a distinct and unique epithelial cell from step b); and d) qualitatively and / or quantitatively assessing cell growth in the clonal culture of step c). According to another embodiment, the method which is the subject of the present invention further comprises the step e) of amplifying the cell population of the clonal culture of step c), or its progeny, by one or more subcultures successive.

It is a question here of producing, from the cell clones obtained at the end of the primary growth step c), independent, parallel, long-term cell cultures via successive subcultures amplified for several weeks. Depending on the requirements, the amplification may be carried out over periods ranging, for example, from approximately 2 to 8 weeks, or even more (see Example of Execution 1, C.2, below). These independent cultures make it possible to obtain a large quantity of cells that can be frozen and stored in the form of one or more cell banks for later use.

The clonal cell libraries that can thus be obtained also represent an object of the present invention. These banks are distinguished from existing banks by the fact that they integrate clonal cell cultures that give them specific structural and functional properties.

In another embodiment, additional or alternative, the method according to the invention further comprises the step f) of evaluating the tissue reconstruction potential of the cell population of the clonal culture of step c), or of his offspring. More precisely, step f) preferably consists in using the cell population of the clonal culture of step c), or its progeny, to reconstruct a three-dimensional tissue so as to evaluate its tissue reconstruction potential. In practice, to carry out this step, cells derived from the primary clonal cultures or micro-cultures can be taken off their culture support and then used individually for each clone of interest to produce a three-dimensional organotypic culture model ( for example, an epithelium, epidermis or reconstructed skin).

The three-dimensional tissues reconstructed from the clonal cultures that can be obtained at the end of step f) of the process according to the invention form part of the objects of the present invention. These tissues are produced according to a new three-dimensional organotypic model because the structural and functional characteristics of the tissue objects of the invention are quite specific insofar as they derive from the properties of a single cell. Such tissues are in particular chosen from the various epithelial tissues, the skin and the epidermis.

In addition, biochips comprising at least one tissue as described above constitute another object of the invention. These biochips can for example be formed from micro-cultures made within a three-dimensional gel made of a biomaterial compatible with cell growth. It is also possible to envisage systems based on multiple reconstructed three-dimensional micro-tissues, each generated independently directly within the biochip, without prior culture step.

In another embodiment, additional or alternative, the method according to the invention further comprises the step g) of evaluating the long-term expansion potential of the cell population of the clonal culture of step c) . More specifically, step g) preferably consists in sub-culturing the cell population of the clonal culture of step c), under conditions promoting cell expansion until the expansion potential is exhausted, so as to evaluate the long-term expansion potential of said cell population.

For example, cells derived from primary clonal cultures or micro-cultures can be removed from their culture support and then subcultured under conditions that promote their multiplication, until their multiplication potential is exhausted.

In another embodiment, additional or alternative, the method according to the invention further comprises the step h) of evaluating the clonogenic potential of the progeny of the cell population of the clonal culture of step c). More specifically, step h) preferably consists in evaluating the clonogenic potential of the progeny of the cell population of the clonal culture of step c), using a quantitative test of the clonogenicity in which one carries out strictly clonal secondary cultures and / or low density cultures allowing the growth of individualized colonies.

For this, we can, for example, take off from their culture medium the cells constituting each clone, then perform, for each of them, a quantitative test of clonogenicity. Thus, it will be possible for example to produce strictly clonal secondary cultures and / or low density cultures allowing the growth of individualized colonies.

Essentially, in the context of the present invention, the initial biological sample is a sample of healthy or diseased epithelial tissue, for example obtained by biopsy in a mammal, preferably in humans. In particular, the tissue sample may be selected from the epithelia, for example the interfollicular epidermis of adult or neonatal human skin, the cornea, the mucous membranes, the hair follicles. Samples of diseased epithelial tissue are for example obtained by biopsy of patients with a genetic disease (such as xeroderma pigmentosum, epidermolysis bullosa, etc.), or by cicatricial skin biopsy (especially for burn victims). The diseased epithelial tissues may also be tumor tissues (carcinomas, etc.)

The biological sample may optionally comprise cells derived from the epithelial (in particular keratinopoietic) differentiation of pluripotent stem cells chosen from embryonic, fetal and induced pluripotent stem cells. Examples of cells having an epithelial potential derived from fetal stem cells are: cells of the ectodermal embryonic leaflet, cells of the epithelial tissues, keratinocytes, etc. Cells with epithelial potential from so-called induced pluripotent stem cells (IPS) are generated by reprogramming cells from adult tissue that may be differentiated cells. Advantageously, the epithelial cells directly extracted from the biological sample are healthy or diseased single cells chosen from progenitor cells, stem cells and keratinocytes.

Another object of the present invention relates to a clonal culture system (or a kit comprising such a system) obtained by the implementation of steps a) to c) at least the method according to the invention. These systems have particular properties inherent in the fact that they derive from clonal cultures. The kits may for example be diagnostic kits, evaluation tests for biological activity, toxicity tests, etc. It is clear to those skilled in the art that the terms "test", "kit" and, possibly, "system" may be, depending on the context in which they are used herein, equivalent.

In a particular embodiment, an optimized clonal epithelial cell culture system for evaluating and exploiting the specific properties of a single cell comprises, in the context of the invention, a culture support in which least one Clonal culture is seeded with a single epithelial cell directly extracted from a biological sample of epithelial tissue according to steps a) through c) of the previously described method.

The present invention also relates to the applications of the method and uses of the various means (system, kit, cell bank, tissue, biochip) described above.

Preferred examples of such uses and applications are:

to identify and select agents having a biological activity of interest. An "agent" can be a candidate molecule that is tested for its biological activity and that is selected according to the applications, said activity being able to be positive (eg, for the selection of effectors of pharmaceutical interest, therapeutic, cosmetic, etc.) or negative (eg, for the selection of toxic molecules). Alternatively, an "agent" may be of a non-chemical nature, for example UV rays, visible light, ionizing radiation, magnetic waves, etc. ;

for treatment and / or diagnosis (especially in vitro) and / or prognosis (especially in vitro) in the field of cell and / or gene therapy, in particular in the field of grafts; to study the specific behavior and / or structural and / or functional properties of a single cell, a cellular subtype, a cell subpopulation, or a cell population;

to produce one or more functional genomic tools, which are particularly useful for inducing phenomena of gain or loss of biological activity, for medical purposes and / or in any type of functional exploration. Examples include interfering RNAs, overexpression and / or repression vectors, viral or non-viral, and the like. ;

to evaluate the efficacy of agents having a biological activity such as molecules of pharmaceutical or cosmetic interest, and / or to evaluate the effectiveness of treatments using such agents (molecules or other types of stimuli, for example waves, light, radiation, physical parameters, etc.).

Finally, the various objects of the present invention have, in relation to the currently available means and methods, the following considerable advantages: (i) the possibility of producing series of parallel micro-cultures, initiated from a single cell, This makes it possible in practice to implement large-scale testing campaigns targeting rare sub-populations, which are poorly represented in tissues, which is not possible in conventional models that consume a lot of cellular material.

(ii) The possibility of initiating clonal cultures from selected phenotype cells seeded individually, for example in microwells, immediately after selection from the tissue, which makes it possible to envisage the implementation of test strategies on a cell material that has not undergone a multiplication step in culture, and therefore less likely to have been modified by artificial culture parameters before testing, especially when the treatment or stimulus is applied immediately after seeding microcultures.

(iii) having, for the first time, access to the behavior of cells placed in culture individually, which offers the possibility of qualifying and quantifying the biological properties and the specific responses of the different cells constituting a population of interest, allowing the analysis of cell heterogeneity, which is not possible in conventional models using globally cell populations.

Thus, the various objects of the present invention find utility in many fields. In addition to the examples of applications already described above, mention may be made, in a nonlimiting manner:

(i) Evaluation of the efficacy of agents with biological activity: gains in function. • Evaluation of the effectiveness of compounds carrying a beneficial biological activity (for example, molecules of pharmaceutical interest, cosmetic active agents):

- Realization of screening campaigns to quantify the effects of biological assets on the scale of a single isolated cell: action of a treatment on the target cell itself, impact on its offspring.

- Opportunity to develop targeted assessment testing strategies for poorly represented populations, such as normal or pathological stem cells.

• Examples: - test effectors likely 1) to induce multiplication of stem cells or progenitor of pluristratified epithelial tissues, 2) to promote maintenance of the stump character in culture in the offspring of isolated stem cells;

- testing of new anti-cancer molecules on stem cells isolated from carcinomas.

(ii) Toxicology issues: loss of function, cancerous transformation.

• Estimation of the impact of stress and toxins at the level of cells treated in isolation, after selection on the basis of precise criteria. These tests can be applied selectively to cells responsible for long-term (stem cell) and short-term (progenitor) renewal of epithelial tissues.

• They also allow, depending on the type of targeted cells, to design tests adapted to the prognosis and distinction of acute or late deleterious effects on a tissue or organ: - Conducting toxicological tests to estimate the safety of a treatment or, on the contrary, to quantify its toxic effects: short-term effect on the isolated cell itself, consequences on its progeny.

- Evaluation of the impact of genotoxic attacks: at the level of cells studied in isolation, DNA damage acquisitions, transmission of mutations and / or genetic anomalies to the offspring, consequences on organogenesis, etc.

(iii) Biochip technology: parallel / massively parallel models of quantification and qualification of biological responses. - Functional genomic screens on two-dimensional cell cultures and / or three-dimensional organotypic models.

- High-throughput screening of molecules carrying a biological activity / highlighting of high-throughput screening ("HTS"). (iv) Cell and gene therapy.

• Qualification of cell samples intended to be grafted, and / or intended to be used for the production of reconstructed tissue grafts:

- Culture tests for estimating the regenerative potential of cells intended for clinical use: growth potential estimated individually on cells under clonal conditions.

Prognostic tests of the engraftment capacity of tissues reconstructed in vitro: estimation of the maintenance or loss of the growth potential of the cells used for the production of grafts. • Validation of gene transfer protocols for clinical purposes:

- Evaluation of the effectiveness of a genetic correction protocol: estimation of the frequency of cells effectively corrected at the end of the gene transfer process.

- Evaluation of the stability of the genetic correction: transmission to the offspring of individualized cells. (v) Cellular and tissue engineering.

Cellular systems and organotypic models compatible with the implementation of isolated cell tests and their offspring:

- Parallel production of cell banks, each constituted by the offspring of a single cell placed in isolation culture.

- In vitro reconstructed tissue models, normal or pathological, generated from the offspring of a single cell placed in culture under clonal conditions.

The following figures illustrate, in connection with the examples below, embodiments of the present invention:

- Figure 1: diagram showing an embodiment of the method according to the invention;

FIG. 2: graphical representation of the results of a long-term expansion experiment for the production of multiple keratinocyte libraries, each resulting from the progeny of a single cell;

- Figure 3: results of an experiment to produce multiple reconstructed epidermis, each derived from the offspring of a single cell; - Figure 4: results of the evaluation of short-term clonal growth of Itgoc6 basal keratinocytes ^strongly placed in culture individually;

FIG. 5: results of an experiment in which the long-term cultures initiated from ^strong Itgoc6 basal keratinocytes, which were placed in culture individually, were quantified;

FIG. 6: graphical representation of the results of an experiment in which the impact of irradiation on epidermal keratinocytes of distinct phenotypes was quantified on the scale of the single isolated cell; FIG. 7: results of an experiment in which the functional test of parallel clonal micro-cultures was used to evaluate the consequences of an irradiation carried out on an isolated cell, on the growth potential of its offspring, and in which we compared the behavior of epidermal keratinocytes of distinct phenotypes;

FIGS. 8A, 8B, 8C: result of a search for chromosome 10 abnormalities by CGH chips: o Descendance of two non-irradiated keratinocytes: K1 and K2 o Descendancy of an irradiated keratinocyte: K3.

Particular embodiments and advantages of the present invention are described in the embodiments below, which relate to the epidermal keratinocytes in ^adult human terfolliculaire.

These examples are provided for illustrative purposes only; they in no way limit the object and scope of the invention.

EXAMPLES

A- EXPERIMENTAL PROCEDURES

A-1 - Preparation of cellular material

Epithelial Cells for Use in Clonal Cultures Tissue biopsies, which are in the examples described herein, of adult human skin biopsies, were first decontaminated, for example by dipping into a skin. physiological solution containing betadine. In order to allow separation between the epithelial tissue and the associated connective tissue (in this case, epidermis and dermis), the samples were then incubated in an enzyme solution at 4 ° C for 10 to 15 hours (Gibco trypsin). At the end of this stage of

SUBSTITUTE SHEET (RULE 26) Enzymatic digestion, the tissue samples were dissected using fine forceps, so as to isolate the epithelial part of the tissue (here, the interfollicular epidermis). The enzymatic treatment, completed by a step of mechanical dissociation by aspiration and discharge using a pipette, extracts the keratinocytes that make up the fragments of epithelia. The cell suspension was finally filtered through a sieve of 50-70 micron mesh size (BD Falcon) to remove cell aggregates. At the end of these steps, the cell samples are in the form of single-cell suspensions, which can be used for seeding clonal cultures.

A- 1-2- Fibroblasts used as support cells

In the examples described, the epithelial cells (keratinocytes obtained from interfollicular epidermis) were studied under clonal conditions in a culture system using as a support a feeder layer of fibroblasts rendered incapable of multiplication by gamma irradiation at a dose of 60 Grays. Thus, these cells remained static but alive, and they supported the growth of the epithelial cells studied. These fibroblasts can in particular be extracted from the dermal part of cutaneous biopsies. For this purpose, dermal fragments were incubated in an enzymatic solution consisting of a mixture of dispase (Roche) and collagenase (Roche) for 2 to 4 hours at 37 ° C. Enzyme digestion, combined with mechanical agitation, allows the extraction of fibroblasts. After removal of the undigested tissue fragments by sieving (BD Falcon) and washing, the resulting fibroblasts were cultured in a medium composed of 90% DMEM (Gibco) and 10% original serum. bovine (Gibco), to be amplified. After multiplication in culture, the fibroblasts were irradiated and then frozen to be stored until use. A-2- Immuno-phenotypic markings

The epithelial cells used to illustrate certain embodiments of the invention are keratinocytes with a high level of expression of the α6 integrin (Itgaβ or CD49ή and low level of expression of the transferrin receptor (CD71): Itga6 phenotype ^{Box faιble} CD71. to carry out the labeling with fluorescent antibody necessary for the definition of this phenotype, cell samples were placed in suspension in physiological saline buffer (PBS) supplemented with 2% bovine serum albumin ( SAB) (Sigma), then incubated for 10 minutes at 4 ° C with mouse immunoglobulins (Jackson Immuno-Research), in order to saturate the nonspecific sites of antibody binding .The labeling of CD49f and CD71 antigens was then carried out by addition of specific antibodies coupled to fluorochromes, then incubation for 30 minutes at 4 ° C: anti-CD49f-PE (phycoerythrin) (clone GoH3, BD Pharmi ngen) and anti-CD71-APC (allophycocyanin) (clone M-A712, BD Pharmingen). After washing the excess antibodies, the samples were ready to be used for seeding the clonal cultures.

A-3 Automated seeding of micro-cultures clonal cultures

/ 4-3-7- Seeding

In the exemplary embodiments described, the keratinocyte clonal micro-cultures were inoculated in an automated manner using a flow cytometer equipped with a cloning module (MoFIo, Cytomation). The excitation of the fluorochromes coupled to the labeling antibodies was performed using a 488 nm argon laser (Coherent) and a 630 nm laser diode. The signals emitted by phycoerythrin (PE) and allophycocyanin (APC) were respectively detected and quantified in wavelength windows of 580 ± 30 nm and 670 ± 30 nm. The sorting criterion chosen in this case corresponded to keratinocytes having the ITGA6 ^{strong faιble} CD71 phenotype and representative of the order of 1% of total keratinocytes: keratinocytes subpopulation described as being enriched in epidermal stem cells (Li et al. , 1998).

A-3-2- Quality control of clonal seeding

In parallel with the clonal cultures intended to be used for the tests and studies (culture conditions described hereinafter), series of clonal seeding were performed in order to validate the cell deposition procedure. These deposits were made under strictly identical technical conditions, but in an environment more favorable to the identification of cells than that used for their growth. This observation medium may be, for example, a physiological saline buffer (PBS) supplemented with Hoeschst 33342 (Sigma) at a concentration of 10 micrograms / ml. Hoechst is a DNA dye that fluoresces under UV excitation. Under these conditions, the cells seeded individually in a large number of culture wells can be identified, which makes it possible to check the efficiency of the process. Each micro-culture must contain only one cell, never two, and the frequency of the empty wells must be minimized.

A-4- Culture conditions

In the examples described, clonal micro-cultures of keratinocytes were made in culture plates comprising 96 wells in which type I collagen (Biocoat, Becton-Dickinson) was adsorbed. The day before seeding the epithelial cells under clonal conditions, a feeder layer of irradiated fibroblasts was placed in the culture wells. These support cells were seeded at a density of 6000 cells / cm ² . The culture medium used for the growth of keratinocytes was based on a mixture of DMEM medium (Gibco) and Ham F12 medium (Gibco), supplemented with serum of bovine origin (Hyclone). This basal medium was in particular supplemented with EGF (Chemicon), insulin (Sigma), hydrocortisone (Sigma), adenine (Sigma), triiodothyronine (Sigma), L -glutamine (Gibco), and with a solution of antibiotics and antimycotics (Gibco). After automated seeding of the keratinocytes at the rate of a single cell per well, the cultures were kept in culture at 37 ° C. in an atmosphere comprising 90% humidity, in the presence of 5% of CO2, in this case for 2 weeks. At the time chosen, a count of the wells in which a cell clone had grown was performed, and then the number of keratinocytes constituting each clone was determined individually after trypsinization of the cells (Gibco).

A-4-2- Study of the tissue reconstruction potential

In the examples described, the tissue reconstruction potential of the progeny of keratinocytes initially placed in culture individually has been demonstrated in an epidermal reconstruction model on dead human dermis epidermis (Régnier et al., 1986). For the preparation of the dermal carriers, human skin samples were incubated for 10 days at 37 ° C in PBS buffer, in order to detach the epidermis, which was removed. Dermis samples without epidermis were cut into squares of about 1 cm ² . They then underwent several successive freezing / thawing cycles which led to the killing of the dermal cells. The acellular dermas obtained were stored at -20 ^° C. until use. The process of reconstructing a three-dimensional epidermis consisted of two stages of successive cultures. The cell samples from the clonal micro-cultures were first seeded on the dermal supports and cultured for 1 week in immersion in a comparable culture medium. similar to that used for primary clonal culture (example of composition described above). The second step of the epidermal reconstruction process was to place the epidermis in formation at the interface between the liquid medium and the ambient air of the incubator. The culture was then continued for 1 to 2 weeks to achieve complete differentiation. The histological features of the reconstructed three-dimensional tissues were visualized after fixation, cutting, and hemalum-eosin-saffron (HES) staining.

A-4-3- Evaluation of Long Term Expansion Capacity

The keratinocytes derived from the clonal micro-cultures were removed by trypsination (Gibco) and then placed in mass culture, individually for each clone studied. These cultures were carried out on plastic surfaces on which type I collagen was adsorbed (for example Petri dishes, Biocoat, Becton-Dickinson). The culture conditions were equivalent to those used for primary clonal growth: feeder layer of irradiated fibroblasts, culture medium of similar composition. After one week, the cultures reached 50% to 80% confluence. The keratinocytes were then removed by trypsination, counted and then reseeded at a density of 2000 to 3000 cells / cm ² , under identical conditions. The cultures were thus transplanted and subcultured each week, until the cell's expansion potential was exhausted. At the end of the stage of primary clone growth and successive subcultures, cell proliferation was estimated in terms of coefficient of expansion and number of population doublings performed, in order to quantify the potential for total expansion of the keratinocytes initially placed in culture individually. Number of doublings of population = (Log N / N ₀ ) / Log 2 N ₀ = Number of cells seeded

N = Number of cells obtained at the end of the culture step. A-4-4- Analysis of secondary clonogenic capacity

It is a question of estimating the maintenance or the loss of the potential to generate colonies among the cells constituting the progeny of the cloned keratinocytes. To do this, the cells of the primary clones were removed by trypsinization (Gibco), then seeded at low density so as to obtain growth of colonies separated from each other (for example, 5 cells / cm ² ) under similar conditions. those described above for the assessment of the long-term expansion potential. After 14 days, the cultures were fixed with 70% ethanol, dried and then stained with two successive baths in eosin (RAL reagents) and RAL Blue 555 (RAL reagents). The parameters taken into account to quantify the clonogenic capacity of the cells studied were in particular the number and the size of the colonies obtained.

B-PERFORMANCE OF THE MEANS OF THE INVENTION A parameter used to analyze the growth potential of epithelial cells is their ability to generate colony-forming efficiency (CFE) colonies or clones when they are present. is a single-celled (CFE) crop. It is obvious that the more methods will demonstrate high CFE values, the more useful they will be for efficiently quantifying the growth potential of epithelial cells. Table I below presents the results obtained according to two known methods of keratinocyte selection and culture (they are conventionally used in the laboratory and have been described in publications), as well as the results obtained according to the method of the invention.

Table I

^* CFE =% of cells capable of giving birth to a colony (low density culture situation) or to a clone (single cell cultures per well). * ^* Average calculated on 3 experiments carried out from independent samples. * ^** Non-exclusively.

C-EXAMPLE 1 Implementation of the model of parallel clonal micro-cultures for the production of multiple keratinocyte libraries, each resulting from the progeny of a single cell (FIG. 2).

C-1- Materials and methods - Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension. - Marking of keratinocytes in suspension using antibodies coupled to fluorochromes making it possible to sort a "stem" cell phenotype (high level of expression of the α6 (Itgαβ) integrin and weak level of expression of the transferrin receptor (CD71): Itga6 ^strong phenotype CD71 ^weak ; selection model: Li et al., 1998).

- Using a flow cytometry cloning module, automated seeding of multi-well culture plates, with a single "strain" phenotype cell per well.

After 2 weeks of culture, identification of the wells in which the cloned keratinocyte gave rise to a cell clone.

- Inoculation of mass cultures from each clonal micro-culture, under conditions favoring cell multiplication. Successive replicating of the cultures in order to obtain the progeny of the cloned initial cells at different stages of amplification (for example: "young" or "aged" culture state), and a quantity of cell material derived from the cloned cells compatible with the constitution of frozen cell banks. - Test of the maximum number of successive subcultures that could be carried out for each culture initiated from a single keratinocyte and estimate the cumulative total number of keratinocytes produced at the end of the long-term cultures.

C-2- Results

On a cohort of 5 cell clones tested for their ability to generate a keratinocyte library, all could be subcultured and their offspring sufficiently amplified to be frozen.

Two weeks after the initiation of the cultures, the 5 clones selected consisted of 8.64 × 10 ⁴ to 1.1 × 10 ⁵ keratinocytes, which is equivalent to 16.40 to 16.86 successive cell generations made since the cloned single cell stage. .

- 1 clone generated a cumulative progeny of 8.48x10 ¹² keratinocytes in 8 weeks of multiplication in culture (No. 1), which is equivalent to a cumulative average population of 42.95 doublings. - 2 clones generated a cumulative progeny of 6.36x10 ¹¹ and 2.4x10 ¹¹ keratinocytes (No. 2 and No. 3), which equates to 39.21 and 37.80 mean doublings of population, respectively.

- 1 clone generated cumulative progeny of 2.03x10 ¹⁰ keratinocytes in 6 weeks of multiplication in culture (No. 4), which is equivalent to a cumulative 34.24 average population doublings. - 1 clone generated a cumulative progeny of 2.15x10 ⁹ keratinocytes in 6 weeks of multiplication in culture (No. 5), which equates to a cumulative 31, 00 average population doublings.

C-3 Conclusion

The model of parallel clonal micro-cultures in accordance with the present invention represents a technology making it possible to generate, in a standardized manner, multiple keratinocyte libraries, each derived from the offspring of a single cell directly isolated from a tissue sample.

It also makes it possible to estimate and compare, at the level of the individual cell, the proliferation potential of distinct cell types (for example: progenitors, epidermal stem cells).

D- EXEMPLARY EMBODIMENT 2

Use of the clonal micro-culture system for the production of multiple reconstructed epidermis, each derived from the progeny of a single cell (Figure 3).

D-1- Materials and methods

- Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension.

- Marking of keratinocytes in suspension using antibodies coupled to flurochromes making it possible to sort in flow cytometry a phenotype of "stem" cells (high level of expression of α6 integrin (Itgaβ) and low level of expression of transferrin receptor (CD71): ^strong Itga6 phenotype CD71 ^weak , selection model: Li et al., 1998 ).

- Using a flow cytometry cloning module, automated seeding of multi-well culture plates, with a single "strain" phenotype cell per well.

After 2 weeks of culture, identification of the wells in which the cloned keratinocyte gave rise to a cell clone.

- Use of the keratinocytes constituting the cell clones, separately for each of them, for the production of reconstructed epidermis (for example: reconstruction of an epidermis on dead human dermis de-epidermis: Regnier et al., 1986).

D-2 Results A cohort of 5 cell clones was tested for the individual ability of each clone to generate a reconstructed three-dimensional epidermis.

The experiment was carried out at a stage of growth of the clones equivalent to that described in the embodiment example No. 1 (multiplication corresponding to -16 to 17 successive cell generations).

The 5 clones tested were found to be capable of producing an epidermis having an organization representative of that of a native epidermis.

D-3- Conclusion

The technology of parallel clonal micro-cultures according to the invention makes it possible to produce reconstructed series of epidermis whose particularity is to be each derived from the offspring of a single cell, whereas the models conventionally used for campaigns large scale tests are generated from banks derived from a mixture of cells.

The reconstructed epidermis produced according to the method that is the subject of the present invention is generated from a cloned cell immediately after extraction from the tissue, and not after a multiplication step in culture, capable of modifying its characteristics.

In this exemplary embodiment, the clones were used for epidermal reconstruction at a growth stage corresponding to -16 to 17 successive cell generations. In other embodiments, epidermal reconstructions may be performed from an earlier or later stage of growth of the cloned cells.

The culture model according to the present invention thus provides an original functional test for qualifying the organogenesis potential of initially cultured cells individually, immediately following selection from the tissue. It offers the possibility to evaluate the impact of a stimulus or stress at different stages of growth of cells placed in culture in isolation, and to study the consequences on the capacity for tissue reconstruction in the short or medium term after treatment.

E-EXEMPLARY EMBODIMENT No. 3

Use of the parallel clonal micro-culture model to characterize the clonogenic capacity of keratinocytes present at a tissue location of interest. In particular, it is necessary to estimate their ability to give birth to a cell clone, the size of which is an indicator of their short-term multiplication potential (Figure 4).

E- 1 - Materials and methods - Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension.

Marking of keratinocytes in suspension using an antibody coupled to a fluorochrome making it possible to sort a population of keratinocytes corresponding to the basal layer of the epidermis (high level of expression of the α6 integrin (Itgaβ): phenotype Itga6 ^strong ).

- Using the flow cytometry cloning module, automated seeding of multi-well culture plates, using a single basal keratinocyte per well.

After 2 weeks of culture, identification of the wells in which the cloned keratinocyte gave rise to a cell clone and then detachment and counting of the keratinocytes, individually for each clone. - Classification of different clones according to their individual size.

E- 2 - Results

The analysis of the clone size distribution generated by keratinocytes derived from the basal layer of the epidermis, performed on a cumulative cohort of -800 clones, reveals the functional heterogeneity of this cell compartment (FIG. 4).

At the top of this hierarchy, there is a minority fraction of clones characterized by an important short-term proliferation capacity, whose size may exceed 15x10 ⁴ keratinocytes in 2 weeks.

- At the bottom of the hierarchy is a fraction of clones characterized by a very limited proliferation capacity. The size of these abortive clones does not exceed 10 ⁴ keratinocytes after 2 weeks of culture. - Between these extremes, is the majority of the clones, which appear distributed along a size gradient, the size value represented mainly being located around 9x10 ⁴ keratinocytes.

E- 3 - Conclusion

The technology of parallel clonal micro-cultures according to the invention makes it possible to precisely estimate the individual clonogenic capacity of the cells of a sample of interest, in this exemplary embodiment a preparation of basal keratinocytes of the epidermis. In particular, the method is resolutive in defining a clonal growth profile providing a representative functional signature of a tissue, or a sub-localization within a tissue, in this case the basal layer of tissue. the adult human interfollicular epidermis. In addition, the system makes it possible to distinguish, within a cell sample of interest, cells having distinct potentialities, as a function of their clonal growth capacity in the short term.

The culture model according to the present invention thus provides an original functional test for estimating the clonogenic capacity of cohorts of cells placed in culture individually. One possible application is the development of quality checks carried out at the individual cell scale to evaluate the functionality (or non-functionality) of cellular samples of interest, compared to a validated reference. The clonal micro-culture system further provides the ability to generate cell samples from a single cell, each individually corresponding to a precisely defined short-term proliferation capability. Another possible application is to use the system for conducting studies to analyze the short-term functional consequences of a treatment (beneficial or toxic) applied at the individual cell level. F-Exemplary Embodiment No. 4

Use of the parallel clonal micro-culture model to characterize the long-term growth potential of keratinocytes of a sample of interest. In particular, it involves detecting the presence of keratinocytes having one of the functional properties associated with epidermal stem cells, namely the ability to perform at least 100 population doublings in culture (FIG. 5).

F- 1 - Materials and methods

- Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension. - Marking of keratinocytes in suspension using an antibody coupled to a fluorochrome allowing to sort a population of keratinocytes corresponding to the basal layer of the epidermis (high level of expression of the integrin α6 {Itgaβ): phenotype Itga6 ^strong ).

- Using the flow cytometry cloning module, automated seeding of multi-well culture plates, with a single basal keratinocyte per well.

After 2 weeks of culture, identification of the wells in which the cloned keratinocyte gave rise to a cell clone.

- Inoculation of mass cultures from a representative cohort of clonal micro-cultures, under conditions favoring cell multiplication.

- Successive replantings of the different crops separately, each week, until reaching the limit of their individual multiplication potential. - Evaluation of the number of successive subcultures that each culture of clonal origin was able to support and estimate the cumulative total number of population doublings performed at each transplant and at the end of the long-term cultures.

F- 2 - Results

The analysis of the long-term growth potential of a cohort of 23 clones derived from basal keratinocytes reveals the marked potential heterogeneity of this compartment (Figure 5). - At the bottom of the observed potential hierarchy are clones with limited growth potential that can only be maintained in culture for 6-7 weeks and only able to perform a total of 30-40 population doublings.

- At the top of the potential hierarchy are clones with a very high growth potential, which can be subcultured for more than 24 weeks without significant reduction in their growth capacity and thus able to exceed the level of growth of 100 doublings of population.

- Between these extremes, are clones distributed according to a wide potential gradient, which can be subcultured for 9 to 18 weeks, and mostly capable of performing 40 to 80 doublings of population.

F- 3 - Conclusion

The parallel clonal micro-culture technology according to the invention makes it possible to precisely estimate the extent of the long-term growth potential of the cells of a sample of interest, in this exemplary embodiment a preparation of basal keratinocytes of 'epidermis. - In particular, the method is resolutive to distinguish a posteriori clones generated from a stem cell, thanks to their ability to accumulate at least 100 population doublings, clones from a progenitor cell, whose long-term proliferation capacity is more limited, usually between 30 and 80 population doublings.

The system makes it possible to generate cell samples corresponding to the progeny of stem cells and progenitor cells, the properties of which can be studied and compared with different phases of their long-term proliferation.

The culture model according to the present invention thus provides an original functional test for qualifying the long-term growth potential of cells initially placed in culture individually. For example, it offers the possibility of estimating the regenerative potential of a sample, in particular by evaluating the presence (or absence) of stem cells. One possible use is to conduct studies to analyze the long-term functional consequences of a treatment (beneficial or toxic) applied at the individual cell level.

G- EXEMPLARY EMBODIMENT 5

Using the functional test of parallel clonal micro-cultures to quantify on the scale of the isolated single cell the impact of irradiation on epidermal keratinocytes of distinct phenotypes (Figure 6).

G-1 - Materials and Methods

- Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension.

- Marking of keratinocytes in suspension using antibodies coupled to flurochromes making it possible to sort in flow cytometry a phenotype of "stem" cells (high level of expression of the α6 integrin (Itgaβ) and low level of expression of the transferrin receptor (CD71): Itga6 ^strong phenotype CD71 ^faιblθ ) and a "progenitor cell" phenotype (high level of integrin α6 {Itgaβ) expression and high level of expression of the receptor transferrin (CD71): ITGA6 ^{strong strong} CD71 phenotype) (selection model: Li et al, 1998)..

- Using a flow cytometry cloning module, automated seeding of multi-well culture plates, at the rate of a single cell per well, and this, for each of the two cellular phenotypes studied.

- Twenty hours after seeding, irradiation of each isolated cell, at a single dose of 2 Gy (γ radiation).

After 2 weeks of culture, counting wells in which a cell clone had grown, then detachment and counts of keratinocytes, individually for each clone.

G-2- Results

G-2-1- Impact of irradiation on the clonogenic capacity of isolated keratinocytes (Figure 6A)

In control condition (without irradiation), the two keratinocyte phenotypes tested showed a strong ability to generate a cell clone. They did not differ significantly on this criterion.

70.3% of the ^strong phenotype Itgα6 phenotype keratinocytes CD71 ^faιblθ (seed cell enriched subpopulation) seeded individually gave rise to a cell clone.

- 61, 7% of Itgα6 keratinocyte phenotype ^{strong strong} CD71 (subpopulation consisted of progenitors) generated a clone.

On the other hand, the keratinocytes of both phenotypes were affected differently by irradiation.

The percentage of keratinocytes with a ^strong CD71 ^Itgα6 phenotype giving rise to a clone was lowered by 70.3% (control condition) at 47.3% (irradiation 2 Gy), which made it possible to estimate the maintenance of the clonogenic capacity of these cells at 67.3%.

- The clonogenic capacity of ITGA6 ^{strong strong} CD71 phenotype of keratinocytes was decreased more drastically by the irradiation: 61, 7% of cells giving birth to a clone in control condition versus 23.0% in conditions of radiation, which estimated a maintenance of clonogenic capacity at only 37.3%.

G-2-2- Impact of irradiation on the growth potential of clones (Figure 6B)

In controlled conditions, the size of the clones produced also did not prove to be a criterion making it possible to clearly distinguish the two types of keratinocytes studied.

The keratinocytes of the two phenotypes have been shown to be capable of generating a large proportion of large clones, comprising at least 5 × 10 ⁴ keratinocytes.

The analysis of the size distributions of the clones obtained provided parameters making it possible to clearly distinguish the specific irradiation responses of the keratinocytes from distinct phenotypes. - Moderate reduction of the capacity of Itgα6 ^strong keratinocytes CD71 ^weak clones to generate large clones following irradiation (median size of the clones in control condition: 10.1x10 ⁴ cells / clone, median size for the irradiated group: 6 8x10 ⁴ cells / clone).

- strongly marked reduction of the capacity of keratinocytes Itgα6 ^{very strong} CD71 producing clones large following irradiation (median size of clones, control group: 8,5x10 ⁴ cells / clone; median size for the irradiated group: 1 2x10 ⁴ cells / clone).

G-3 Conclusion The model of parallel clonal micro-cultures according to the invention is efficient for analyzing the growth capacity of keratinocytes of specific phenotypes at the level of the individual cell. Indeed, the clonogenic efficiency values obtained in this model, reaching 60 to 70% of the cloned cells, prove to be much higher than what is generally described in conventional culture systems, concerning keratinocytes directly derived from a biopsy of tissue, for which the values are of the order of 10%.

- This model is also effective to detect, qualify and quantify a deleterious effect on the cell growth potential. This example illustrates the ability of the system to be enhanced by the development of in vitro tests of radio-toxicology.

EXAMPLE 6 Use of the functional test of parallel clonal micro-cultures to evaluate the consequences of the irradiation of a single cell on the growth potential of its offspring. Comparison of the behavior of epidermal keratinocytes of distinct phenotypes (Figure 7).

H-1- Materials and methods

Following the procedure described in the context of the embodiment example 5:

- Low density seeding of a portion of keratinocytes from clones, so as to obtain individualized colonies: in this example, density of 5 keratinocytes / cm ² .

- After 2 weeks of culture, fixing and staining colonies, so as to make a microscopic and macroscopic observation.

H-2- Results In control conditions (without irradiation of the initial cells placed in culture individually), the groups of clones tested for their ability to generate secondary colonies showed very similar characteristics for this criterion. - The ability to produce secondary colonies of the two series of clones, respectively from ITGA6 ^strong CD71 ^faιblθ keratinocytes [clones (1) to (5)] and keratinocytes ITGA6 ^{strong strong} CD71 [clones (11) to (15)], proved to be comparable.

These 2 groups included both cell clones giving abundant birth to large secondary colonies [eg clones (1) and (11)] and clones giving rise to small and small colonies [ for example: clones (5) and (15)].

With respect to clone groups from an irradiated cell (single dose 2 Gy), the ability to generate secondary colonies was clearly distinct for the 2 keratinocyte phenotypes tested.

The group of clones derived from Itga6 ^strong CD71 ^weak keratinocytes [clones (6) to (10)] showed a capacity to produce secondary colonies equivalent to that of non-irradiated groups.

- In contrast, the group of clones from ITGA6 ^{strong strong} CD71 keratinocytes undergoing irradiation [clones (16) to (20)] showed a decreased secondary clonogenic capacity (loss of colonies of diameter> 5 mm).

H-3- Conclusion

The model of parallel clonal micro-cultures according to the present invention is adapted to highlight, qualify and quantify the non-immediate consequences of irradiation carried out on keratinocytes studied individually. In this case, the effect deleterious highlighted was a loss of growth capacity measured on the progeny of cells placed in clonal culture.

EXEMPLARY EMBODIMENT No. 7

Use of the parallel clonal micro-culture model to analyze the long-term consequences of genotoxic stress applied to cells placed in clonal conditions. This involves applying stress a few hours after seeding the cells individually into separate culture wells, and then initiating long-term cultures after the cells have split. The presence of abnormalities in the genome is then sought at the level of the progeny of the cloned cells, after it has made a number of population doubling determined. This research is for example carried out using a technique for highlighting the imbalances of representation of DNA sequences in the genome (deletions, amplifications): comparative genomic hybridization (CGH) (Figure 8).

1-1 - Materials and methods

- Extraction of keratinocytes from an epidermis resulting from an adult skin biopsy (breast reduction), dissociation and suspension.

- Marking of keratinocytes in suspension using an antibody coupled to a fluorochrome allowing to sort a population of keratinocytes corresponding to the basal layer of the epidermis (high level of expression of the integrin α6 {Itgaβ): phenotype Itga6 ^strong ).

- Using the flow cytometry cloning module, automated seeding of multi-well culture plates, with a single basal keratinocyte per well. Separation of the clonal micro-cultures in 2 groups: 1) group subjected to a single dose of 2 Grays of gamma radiation 19 hours after seeding; 2) non-irradiated control group.

After 2 weeks of culture, for each of the 2 groups, identification of the wells in which the cloned keratinocyte gave rise to a cell clone.

Seeding of mass cultures from cohorts of clonal micro-cultures representative of each of the two groups, under conditions favoring cell multiplication. - Successive replantings of the different crops separately, each week, until reaching the limit of their individual multiplication potential.

- Evaluation of the number of successive subcultures that each culture of clonal origin was able to support and estimate the cumulative total number of population doublings performed at each transplant and at the end of the long-term cultures.

- Among the control and irradiated groups, selection of cultures showing a long-term proliferation capacity very important, in particular capable of carrying out at least 150 doublings of population.

For each candidate selected, preparation of genomic DNA samples corresponding to a late stage of long-term proliferation, in this case the cultures having made about 150 doublings of population after clonal sowing. - On the DNA samples generated, from the control and irradiated groups, search for genome zones presenting deletion and / or amplification anomalies by comparative genomic hybridization (CGH) versus reference DNA (CGH Constitutional Chip ^® 4.0 chips, PerkinElmer, Inc. according to the method recommended by the manufacturer).

I-2 - Results CGH flea cytogenetic analysis of long-term cultures of clonal origin shows that gamma dose

Grays results in the acquisition of chromosomal abnormalities transmitted to offspring, which are found to be detectable by a large number of cell divisions after application of genotoxic stress (Figure 8).

In the example presented, an amplification of a 44.3 mega-base locus located on chromosome 10 (region 10q11.21-10q23.1) is detected at the level of the DNA of the offspring. a keratinocyte, 150 doublings of population after it has been irradiated.

- In contrast, this same genomic segment studied at the DNA level of the offspring of 2 examples of keratinocytes that have not been irradiated does not exhibit this type of genome alteration.

1-3 - Conclusion

- The clonal micro-culture technology makes it possible to subject individual epidermal basal keratinocytes to genotoxic stress, in this case gamma irradiation, and then to evaluate the long-term consequences on the offspring. - In particular, the method is valid for analysis at the clonal level, the impact of stress on the genome integrity of basal keratinocytes.

In this exemplary embodiment, the system makes it possible to demonstrate a genotoxic effect of gamma irradiation on keratinocytes derived from the basal layer of the epidermis.

The culture model according to the present invention thus provides an original system for performing toxicology tests at the individual cell scale. It offers, for example, the possibility of characterizing the individual sensitivity of basal keratinocytes of the epidermis to genotoxic agents. One possible use is the performance of tests aiming at detecting the occurrence of genome defects such as deletions and / or amplifications, as a result of exposure to a toxin, and analyzing their transmission to the offspring during successive cell divisions, particularly in the long term.

REFERENCES

• Gangatirkar et al., Nat. Protoc. 2007. 2: 178-186

• Li et al., Proc Natl Acad Sci USA. 1998. 95: 3902-3907 Regnier et al., Exp CeII Res. 1986. 165: 63-72

• Lorenz K et al. CeIIs Tissues Organs. 2008 Aug 11.

• Barrandon and Green, Proc Natl Acad Sci USA. 1987. 84: 2302-2306

• Larder et al., Stem CeIIs. 2006. 24: 965-974. Rachidi et al., Radiother Oncol. 2007. 83 (3): 267-76.

Claims

A clonal epithelial cell culture system optimized for evaluating and exploiting specific properties of a single cell, wherein a culture support comprises at least one clonal culture seeded with a single epithelial cell directly extracted from a biological sample of healthy or diseased epithelial tissue.

2. System according to claim 1, characterized in that said culture support comprises at least two parallel clonal cultures, each of said cultures being seeded with a distinct and unique epithelial cell directly extracted from said biological sample.

3. System according to claim 1 or 2, characterized in that it is in the form of biochip.

A method of clonally culturing epithelial cells optimized for evaluating and exploiting specific properties of a single cell, comprising at least the steps of: a) extracting one or more epithelial cells directly from a sample biological healthy or diseased epithelial tissue; b) optionally, selecting at least one population and / or subpopulation of epithelial cells from the cells extracted in step a); c) performing a clonal culture seeded with a distinct and unique epithelial cell directly from step a) or b); and d) qualitatively and / or quantitatively assessing cell growth in the clonal culture of step c).

5. Method according to claim 4, characterized in that it further comprises the step e) of amplifying the cell population clonal culture of step c), or its progeny, by one or more successive subcultures.

6. Method according to claim 4 or 5, characterized in that it further comprises the step f) of using the cell population of the clonal culture of step c), or its progeny, to reconstruct a three-dimensional tissue. , so as to evaluate its potential for tissue reconstruction.

7. Method according to any one of claims 4 to 6, characterized in that it further comprises step g) of subculturing the cell population of the clonal culture of step c) under conditions promoting cell expansion until the expansion potential is exhausted, so as to evaluate the long-term expansion potential of said cell population.

8. Method according to any one of claims 4 to 7, characterized in that it further comprises step h) of evaluating the clonogenic potential of the progeny of the cell population of the clonal culture of step c ), using a quantitative clonogenicity test in which strictly clonal secondary cultures and / or low density cultures are grown to allow individualized colonies to grow.

9. Method according to any one of claims 4 to 8, characterized in that step c) comprises the production of at least two parallel clonal cultures, each of said cultures being seeded with a distinct and unique epithelial cell directly derived from step a) or b).

10. System according to any one of claims 1 to 3, or method according to any one of claims 4 to 9, characterized in that said biological sample of epithelial tissue is obtained by biopsy in a mammal, preferably in the man.

11. System according to any one of claims 1 to 3 and 10, or method according to any one of claims 4 to 10, characterized in that said epithelial tissue is selected from epithelia, for example interfollicular skin epidermis adult or neonatal human, cornea, mucous membranes, hair follicles.

12. System according to any one of claims 1 to 3, 10 and 11, or method according to any one of claims 4 to 11, characterized in that the epithelial cell or cells directly extracted from said biological sample is (are) a healthy single or diseased cell (s) chosen from progenitor cells, stem cells, keratinocytes.

13. System according to any one of claims 1 to 3, characterized in that it is obtained by the implementation of steps a) to c) at least the method of claim 4.

14. Clonal cell bank obtainable at the end of step e) of the process according to claim 5.

15. Three-dimensional fabric reconstructed from a clonal culture that can be obtained at the end of step f) of the method according to claim 6.

16. The fabric as claimed in claim 15, characterized in that it is chosen from the epithelia, the skin and the epidermis.

A biochip comprising at least one tissue according to claim 15 or 16.

18. Use:

the system according to any one of claims 1 to 3 and 10 to 13, or

the cell bank according to claim 14, or

- The tissue of claim 15 or 16, or - the biochip of claim 17, or application of the method according to any one of claims 4 to 12, for identifying and selecting agents having a biological activity of interest.

19. Use:

the system according to any one of claims 1 to 3 and 10 to 13, or

the cell bank according to claim 14, or

tissue according to claim 15 or 16, or the biochip according to claim 17, or application of the method according to any one of claims 4 to 12, for the in vitro diagnosis and / or the prognosis in vitro in the field cellular and / or gene therapy, particularly in the field of grafts.

20. Use:

the system according to any one of claims 1 to 3 and 10 to 13, or

the cell bank according to claim 14, or the tissue of claim 15 or 16, or

the biochip according to claim 17, or application of the method according to any one of claims 4 to 12, to study the behavior and / or the specific structural and / or functional properties of a cell, a cellular subtype, a cell subpopulation , or a cell population.

21. Use:

the system according to any one of claims 1 to 3 and 10 to 13, or

the cell bank according to claim 14, or the tissue of claim 15 or 16, or

the biochip according to claim 17, or the method according to any one of claims 4 to 12, for producing one or more functional genomic tools.

22. Use:

the system according to any one of claims 1 to 3 and 10 to 13, or

the cell bank according to claim 14, or

the tissue according to claim 15 or 16, or the biochip according to claim 17, or applying the method according to any one of claims 4 to 12, to evaluate the efficacy or the impact of treatments, in particular by means of agents with biological activity, stress, toxic, genotoxic attacks.