MICROSCOPIC METHOD FOR CHARACTERIZING EPIDERMAL CELL RENEWAL

Abstract

The present invention relates to a method for the in vitro characterization and quantification of proliferative cells in an epidermal fragment, comprising the following steps: 1) Cultivating, on a support, the epidermal fragment, 2) Detaching the epidermal fragment from the support, 3) Carrying out immunolabeling on the epidermal fragment, 4) Acquiring images of the basal layer of the epidermal fragment over its entire surface, 5) reconstructing an image representing the entire basal layer of the epidermal fragment, 6) Selecting the immunolabeled nuclei of the proliferative cells by image processing, 7) Quantifying cell division by counting the nuclei by image analysis. The present invention also relates to a use of the method for evaluating the effect of a product or an active ingredient on epidermal cell regeneration. Figure 1

Description

Title of the invention: MICROSCOPIC METHOD FOR CHARACTERIZING EPIDERMAL CELL RENEWAL Technical field

[0001] The present invention relates to a method for the in vitro characterization and quantification of proliferative cells in a fragment of epidermis, as well as to its use for evaluating the effect of a product or an active ingredient on epidermal cell regeneration.

[0002] The present invention finds applications in particular in the field of cosmetics and dermatology, in particular in the field of the evaluation of cosmetic and dermatological products. State of the art

[0003] The skin is a vital organ in its own right, consisting of three distinct tissues, each performing different roles thanks to different cell types and structures.

[0004] The most superficial tissue, and therefore the most exposed to external stimuli, is the epidermis. This multi-stratified (Malpighian) and keratinized epithelium is composed of different cells associated with numerous barrier and protection functions. The majority of cells are keratinocytes, which divide in the basal layer and begin their differentiation up to the horny layer (outermost layer), then are eliminated by desquamation, in 21 to 28 days on average. The major role of the epidermis is to provide the skin, and therefore the human body, with a first line of protection against external aggressions, such as physical, chemical, water and bacteriological aggressions. This protection is notably ensured by the most differentiated layers and the horny layer, known for its hydrophobic properties, its compact and waterproof appearance.Good differentiation and therefore good desquamation are essential for a smooth, homogeneous and regular surface. All of these mechanisms depend directly on the proliferative capacities of keratinocytes. However, external stresses, such as UV rays, and age alter keratinocytes and slow down this process, which can then become longer and more irregular. As desquamation becomes irregular and / or anarchic, defects in the smoothing and homogeneity of the skin surface occur.

[0005] For the cosmetic or dermatological market, it is therefore appropriate to prevent or correct these effects on the appearance of the skin, in particular by providing products allowing better hydration, nutrition, and / or protection of the skin, and more overall better epidermal cell renewal. Thus, the market for cosmetic and dermatological products is very large, and consumer expectations are very high.

[0006] Before any marketing of a cosmetic or dermatological product, it is necessary to test its effectiveness, in particular on epidermal cell renewal. For this purpose, it is customary to use in vitro skin models, in particular reconstructed human epidermis (also called "RHE" for Reconstructed Human Epidermis) or skin equivalents. However, the histology techniques conventionally used consist in particular of making cross-sections of RHE, which only allows for partial information.

[0007] Thus, conventional histological techniques, combined with classical microscopic / histological observation techniques, prevent observation of the entire surface of the model.

[0008] There is therefore a real need for a method making it possible to meet these needs and drawbacks of the prior art.

[0009] Description of the invention

[0010] The present invention aims precisely to meet these needs and drawbacks of the prior art.

[0011] The inventors have in fact developed a technique for marking and observing the surface of a skin model as a whole.

[0012] The method of the invention makes it possible to observe the cellular renewal of basal keratinocytes, which are at the heart of epidermal renewal, despite the constraints linked to the use of an epidermal model.

[0013] The inventors of the present invention have in fact developed a method allowing on the one hand a measurement of cell proliferation over the entire surface of the basal layer of the epidermis model, and on the other hand the obtaining of results of measurement of cell proliferation over the entire epidermal model. Advantageously, the results obtained do not result from an extrapolation, as is generally the case in the methods of the prior art, but are indeed the direct result of the implementation of the method of the invention.

[0014] Advantageously, the method of the invention makes it possible to characterize and quantify proliferative cells, also called dividing cells, within an epidermal model. It thus makes it possible to evaluate the cell renewal properties of an epidermal model, in particular in the context of testing a cosmetic or dermatological product or active ingredient.

[0015] Thus, a first object of the invention relates to a method for characterizing and quantifying in vitro proliferative cells in a fragment of epidermis, comprising the following steps:

[0016] 1) Cultivate, on a support, the fragment of epidermis,

[0017] 2) Separate the fragment of epidermis from the support,

[0018] 3) Carry out immunolabeling on the epidermis fragment,

[0019] 4) Acquire images of the basal layer of the epidermis fragment over its entire surface,

[0020] 5) Reconstruct an image representing the entire basal layer of the fragment of epidermis,

[0021] 6) Select the immunolabeled nuclei of proliferating cells by treatment of image, and

[0022] 7) Quantify cell division by counting nuclei by analysis of the image.

[0023] A second subject of the invention relates to the use of the characterization and quantification method of the invention, to evaluate the effect of a product or an active ingredient on epidermal cell regeneration.

[0024] For the purposes of the present invention, the term "characterizing proliferative cells" means visualizing and identifying, using immunolabeling, a proliferation marker present in keratinocytes, and which is expressed only during the division phases of the cell cycle. Characterizing proliferative cells amounts to identifying those which express this marker. The proliferation marker may be any marker known to those skilled in the art. It may, for example, be at least one marker chosen from Ki-67, MCM2, p53, PCNA, Bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (EdU), and AGNOR (AgNucleolar Organizer Regions). Preferably, it may be Ki-67.

[0025] For the purposes of the present invention, the term "epidermal fragment" means any tissue structure having the same structural characteristics, in particular three-dimensional, as those of the superficial layer of the skin which covers the dermis. As such, the epidermal fragment further comprises a basal layer. Advantageously, the epidermal fragment reproduces the behavior of the epidermal layer of the skin. It may, for example, be a reconstructed human epidermis or a skin sample.

[0026] For the purposes of the present invention, the term "proliferative cell" means any healthy cell reproducing by mitosis, that is to say by dividing itself, into a line of healthy cells. A qualitative and / or quantitative level of proliferative cells in the epidermis fragment higher than a reference level of proliferative cells in a healthy epidermis reveals improved cell renewal. Conversely, a qualitative and / or quantitative level of proliferative cells in the epidermis fragment lower than a reference level of proliferative cells in a healthy epidermis reveals degraded cell renewal. The reference level may be the level measured in healthy epidermis, or the control level measured on the epidermis fragment before any treatment of it.

[0027] Step 1) of culturing the epidermis fragment can be carried out according to any suitable method known to those skilled in the art. In this respect, the culture can be carried out in any suitable culture medium and under culture conditions known to those skilled in the art. This includes in particular a medium and culture conditions allowing cell growth and differentiation at a level equivalent to that encountered in vivo. This can be, for example, the SGM+ medium (Episkin™), or any other medium provided and adapted to the epidermis model chosen for the experiment. The duration of the culture can be adapted according to the needs of the experiment.

[0028] The support of the epidermis fragment may be any support known for this use by those skilled in the art. It may be, for example, a polycarbonate membrane or a collagen matrix. The support is in contact with the basal layer of the epidermis fragment.

[0029] Step 2) of detaching the epidermis fragment from the support can be carried out by any suitable method not involving destruction or damage to the epidermis fragment. Preferably, the detachment is carried out at the base of the cells of the basal layer of the epidermis fragment. Advantageously, the entire epidermis fragment is detached from the support at the end of this step. Any known substance allowing detachment can be used, for example a dispase.

[0030] At the end of the detachment step, the epidermis fragment can be preserved so as to avoid any degradation according to a method known to those skilled in the art. For example, the epidermis fragment can be fixed in paraformaldehyde.

[0031] Step 3) of immunolabeling on the epidermis fragment can be carried out according to any method known to those skilled in the art. It may be, for example, direct immunolabeling by antibodies directed against at least one cell proliferation marker, for example chosen from Ki-67, MCM2, p53, PCNA (proliferating cell nuclear antigen), Bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (EdU), and AGNOR (AgNucleolar Organizer Regions). Optionally, the antibody may be coupled to a fluorophore, for example Alexa 488. Otherwise, a secondary antibody coupled to a fluorophore must be used in order to highlight the selected marker. Advantageously, immunolabeling participates, in the subsequent steps of the method, in the visualization and / or selection and / or quantification of the proliferating cells.

[0032] Optionally, a permeabilization step of the detached epidermis fragment can be carried out before immunolabeling, in order to allow the antibodies to penetrate the nucleus of the cells. Advantageously, this allows a more efficient analysis. since the process of keratinocyte cell renewal involves nuclear markers.

[0033] Optionally, a nuclear counterstaining step can be carried out using a nuclear stain, for example Hoechst stain. This step makes it possible to visualize the proportion of proliferative cells among all the cells present in the basal layer of the epidermis. It also provides the possibility of making a proliferative cell / total cell ratio, allowing a more detailed analysis and comparison of the different conditions tested.

[0034] Step 4) of acquiring images of the basal layer of the epidermis fragment over the entire surface thereof can be carried out by any suitable means known to those skilled in the art, for example by means of a fluorescence photonic microscope with motorized stage, equipped with a CCD camera. Advantageously, the images can be mosaic images, for example constructed automatically, advantageously to increase the field of view while preserving the microscopic resolution.

[0035] Steps 5) of reconstructing an image representing the entire basal layer of the epidermis fragment and 6) of selecting the nuclei of the proliferating cells by image processing can be done using any suitable software, for example the FIJI (National Institutes of Health) software. The image processing can be any known processing or a combination of processing, for example thresholding, rectification, normalization, smoothing, frequency analysis and / or source separation analysis. Preferably, thresholding is used.

[0036] Step 7) of quantifying cell division by counting the immunolabeled nuclei by image analysis can be done by any means commonly used for this purpose by those skilled in the art, for example by means of calculation software or visually by semi-automated counting.

[0037] Advantageously, the method of the invention does not include a step of inclusion in paraffin and / or of carrying out histological sections. This is an advantage compared to most of the methods of the state of the art which cannot be exempt from these steps.

[0038] Advantageously, it is possible to treat the epidermal fragment, during step 1) of culturing it, with a product or an active ingredient, for example cosmetic or dermatological, to test the effect of the product or active ingredient on epidermal cell proliferation and / or regeneration in the epidermal fragment by implementing the method of the invention. In this case, it is possible to compare the effect of the product or active ingredient with a control, for example a positive control made with any substance known to cause cell proliferation, such as KGF. (Keratinocyte Growth Factor), and / or to a negative control, for example the untreated epidermal fragment or a reference level measured in healthy epidermis.

[0039] Any product or active ingredient can be tested. A product is a formulation comprising at least one active ingredient, in particular cosmetic or dermatological, and at least one other ingredient.

[0040] The method of the invention can further be used to evaluate an effect, advantageously on the skin, in particular the face, of a product or an active ingredient on epidermal cell regeneration, for example on the signs of aging, the maintenance or improvement of the barrier function of the skin. For example, the effect on the signs of aging can be a resurfacing, smoothing effect, in particular on wrinkles and fine lines and / or plumping.

[0041] Other advantages may also become apparent to those skilled in the art upon reading the examples below, illustrated by the appended figures, given for illustrative purposes. Brief description of the drawings

[0042] [Fig.l] represents the photos of the untreated models, the photos of the models treated with KGF and the photos of the models treated with the anti-aging care product 2 or the anti-aging care product 3 (visualization after immunolabeling, the proliferating cells marked positively by Ki-67 appear in white).

[0043] [Fig.2] represents the quantification of the number of nuclei in renewal. S*: statistically different in the Wilcoxon Mann-Whitney median test - NS: statistically not different in the Wilcoxon Mann-Whitney median test, for the untreated models, the models treated with KGF and the models treated with anti-aging care product 2 or anti-aging care product 3.

[0044] Examples

[0045] Example 1: Evaluation of anti-aging care formula 1 on the renewal of keratinocytes in a reconstructed epidermis by quantification of the cell proliferation marker Ki-67

[0046] The reconstructed human epidermis (“RHE”, Episkin™ RHE / S / 10) is a skin model that can reproduce the characteristics and behavior of the epidermal layer of the skin, and thus allows us to evaluate the effect of an active ingredient or formula on the capacity of keratinocytes to proliferate. This model then allows us to observe the cell renewal of basal keratinocytes which are at the heart of epidermal renewal and thus to demonstrate a beneficial effect of the anti-aging care formula 1. These reconstructed epidermis also have the advantage of being reproducible because they come from characterized lines and have a standardized size of 0.5 cm2 in diameter.

[0047] Reception and preparation of reconstructed epidermis (D-24h):

[0048] After receiving the models 10 days after their manufacture, the RHE medium is changed (SGM+ medium, Episkin™) and then the latter are placed in a cell incubator under controlled conditions (Temperature 37°C, CO2 5%, humid atmosphere) for 24 hours. These steps ensure that the epidermis are in optimal conditions for carrying out the experiment.

[0049] Treatment of reconstructed epidermis:

[0050] The RHE are treated or not (control) the next day (D0) by topical application of the anti-aging care formula 1 at a rate of 2 mg / cm2, i.e. 3 conditions considered during the experiments. Each condition is repeated 8 times (8 replicates): - No treatment corresponding to the untreated control condition (n=8). - anti-aging care formula 1 (n=8). - Conditions where the culture medium contains KGF (K1757-10UG, sigma) at 100 ng / ml (n=8) = positive control

[0051] The treatments and media (SGM+ supplied by Episkin™) are renewed every day (excluding weekends) at a rate of 1 ml per well or 2 mg / cm2 for the anti-aging care formula 1.

[0052] Fixation of RHE (J5), immunostaining:

[0053] The treatment was stopped after 5 days of incubation. The protocol followed to detach the basement membrane from its support and immunolabeling is as follows: - Application of Dispase II ® solution (2.4 IU / mL, Sigma) directly into the well for 2 min at room temperature - Detachment of the basal layer of the polycarbonate membrane using forceps - Fixation with 2% paraformaldehyde for 1 hour - Rinse with PBS 5 min x 2 - Permeabilization of RHE with Triton solution - Saturation of non-specific antigenic sites with BSA solution - Specific labeling with an antibody directed against the Ki-67 protein and coupled to the Alexa 488 fluorophore (Abcam) - Nuclear counterstaining with Hoechst (labeling of nuclei)

[0054] Microscopic characterization:

[0055] The models were characterized in epifluorescence (Leica DM6) with the xl0 fluorescence objective - Leica DAP filters for highlighting the Hoechst labeling and in L5 filter for the Ki-67 labeling.

[0056] The parameters are shown in Table 1.

[0057] [Tables 1] Dye / Label Filter Emission Acquisition Time Hoechst DAP 447 nm 60 ms Ki-67 FITC (L5) 527 nm 365.011 ms

[0058] Quantification:

[0059] The counting of proliferating nuclei (Ki-67 labeled cells) from the mosaic images was carried out using a macro-command (automated sequence of commands allowing the processing and then analysis of the images) on the FIJI image analysis software. Using this macro-command, the entire model is characterized. Automatic thresholding allows the selection of gray levels from 110 to 255.

[0060] A statistical analysis (data not shown) allows us to conclude on the significant increase in the number of proliferating nuclei after treatment with the tested product.

[0061] Results:

[0062] The untreated samples show homogeneity in terms of preparation and marking. The average quantity of cores in renewal on the 8 models is 21225 cores + / - 28%.

[0063] Samples treated with the positive control KFG are homogeneous and appear visually whiter (therefore more marked with KI67) than untreated samples. The number of nuclei in renewal increases significantly compared to untreated models, with an average of 33875 nuclei + / - 32%.

[0064] In the samples treated with the Anti-aging 1 care formula, the number of nuclei in renewal increases significantly in comparison with the untreated models, with an average of 29565 nuclei + / - 35%.

[0065] Conclusion: The specific method developed allows quantification of dividing cells over the entire surface of the reconstructed epidermis. In addition, the anti-aging care formula 1 induces significant cell proliferation after 5 days of treatment compared to untreated RHE models.

[0066] Example 2: Evaluation of two anti-aging care formulas on the renewal of keratinocytes in a reconstructed epidermis by quantification of the cell proliferation marker Ki-67

[0067] The reconstructed human epidermis (“RHE”, Episkin™ RHE / S / 10) is a skin model that can reproduce the characteristics and behavior of the epidermal layer of the skin, and thus makes it possible to evaluate the effect of an active ingredient or formula on the capacity of keratinocytes to proliferate. This model then makes it possible to observe the cell renewal of basal keratinocytes which are at the heart of epidermal renewal and thus to demonstrate a beneficial effect of the treatment tested. These epidermis reconstructed also have the advantage of being reproducible because they come from characterized lines and have a standardized size of 0.5 cm2 in diameter.

[0068] Reception and preparation of reconstructed epidermis (D-24h):

[0069] After receiving the models 10 days after their manufacture, the RHE medium is changed (SGM+ medium, Episkin™) and then the latter are placed in a cell incubator under controlled conditions (Temperature 37°C, CO2 5%, humid atmosphere) for 24 hours. These steps ensure that the epidermis are in optimal conditions for carrying out the experiment.

[0070] Treatment of reconstructed epidermis:

[0071] The RHE are treated or not (control) the next day (D0) by topical application of the two anti-aging treatments at a rate of 2 mg / cm2 each, i.e. 4 conditions considered during the experiments. Each condition is repeated 6 times (6 replicates): - No treatment corresponding to the untreated control condition (n=6). - Anti-aging care formula 2 (n=6). - Anti-aging care formula 3 (n=6). - Conditions where the culture medium contains KGF (K1757-10UG, sigma) at 100 ng / ml (n=6) = positive control

[0072] The treatments and media (SGM+ supplied by Episkin™) are renewed every day (excluding weekends) at a rate of 1 ml per well or 2 mg / cm2 for both formulas.

[0073] Fixation of RHE (J5), immunostaining:

[0074] The treatment was stopped after 5 days of incubation. The protocol followed to detach the basement membrane from its support and immunolabeling is as follows: - Application of Dispase II ® solution (2.4 IU / mL, Sigma) directly into the well for 2 min at room temperature - Detachment of the basal layer of the polycarbonate membrane using forceps - Fixation with 2% paraformaldehyde for 1 hour - Rinse with PBS 5 min x 2 - Permeabilization of RHE with Triton solution - Saturation of non-specific antigenic sites with BSA solution - Specific labeling with an antibody directed against the Ki-67 protein and coupled to the Alexa 488 fluorophore (Abcam) - Nuclear counterstaining with Hoechst (labeling of nuclei)

[0075] Microscopic characterization:

[0076] The models were characterized in epifluorescence (Leica DM6) with the xl0 fluorescence objective - Leica DAP filters for highlighting the Hoechst labeling and in L5 filter for the Ki-67 labeling (see [Fig.l]).

[0077] The parameters are shown in Table 2.

[0078] [Tables2] Dye / marking Filter Emission Acquisition time Hoechst DAP 447 nm 60 ms Ki-67 FITC (L5) 527 nm 365.011 ms

[0079] Quantification:

[0080] The counting of proliferating nuclei (Ki-67) from the mosaic images was carried out using a macro command on the FIJI image analysis software. Using this macro command, the entire model is characterized. Automatic thresholding allows the selection of gray levels from 110 to 255. The quantification results are shown in [Fig.2].

[0081] A statistical analysis (data not shown) allows us to conclude on the significant increase in the number of proliferating nuclei after treatment with the tested product.

[0082] Results:

[0083] The untreated samples show homogeneity in terms of preparation and marking. The average quantity of cores in renewal on the 6 models is 14911 cores + / - 17%.

[0084] Samples treated with the positive control KFG are homogeneous and appear visually whiter than untreated samples. The number of nuclei in renewal increases significantly compared to untreated models, with an average of 55755 nuclei + / - 10%.

[0085] Samples treated with the anti-aging care formula 2 are homogeneous in terms of marking. The number of nuclei in renewal increases very slightly in comparison with the untreated models, with an average of 16,286 nuclei + / -13%. This difference is not significant.

[0086] Samples treated with the anti-aging care formula 3 are homogeneous in terms of marking. The number of nuclei in renewal increases significantly compared to untreated models, with an average of 23,346 nuclei + / -14%.

[0087] Conclusion: The specific method developed makes it possible to quantify dividing cells over the entire surface of the reconstructed epidermis, and to compare the regenerative properties of cosmetic formulas with each other. Thus, the anti-aging care formula 3 induces significant cell proliferation after 5 days of treatment in comparison with untreated RHE models.

Claims

Claims

1. Method for in vitro characterization and quantification of proliferative cells in a fragment of epidermis, comprising the following steps: 1) Cultivating, on a support, the fragment of epidermis, 2) Detaching the fragment of epidermis from the support, 3) Carrying out immunolabeling on the fragment of epidermis, 4) Acquiring images of the basal layer of the fragment of epidermis over its entire surface, 5) reconstructing an image representing the entire basal layer of the fragment of epidermis, 6) Selecting the immunolabeled nuclei of the proliferative cells by image processing, 7) Quantifying cell division by counting the nuclei by image analysis.

2. Method according to claim 1, in which said epidermis fragment is treated, during step 1) of culturing it, with a product or an active ingredient, for example cosmetic or dermatological, to test the effect of the product or active ingredient on cell proliferation in the epidermis fragment.

3. A method according to claim 1 or 2, wherein the epidermis fragment is a reconstructed epidermis or a skin sample.

4. A method according to any preceding claim, wherein said support is selected from a polycarbonate membrane or a collagen matrix.

5. Method according to any one of the preceding claims, in which the immunolabeling is a direct immunolabeling by antibodies directed against at least one cell proliferation marker, for example chosen from Ki-67, MCM2, p53, PCNA, Bromodeoxyuridine (BrdU), 5-ethynyl-2'-deoxyuridine (EdU), and AGNOR (AgNucleolar Organizer Regions), optionally coupled to a fluorophore, for example Alexa-488.

6. Method according to any one of the preceding claims, characterized in that it does not include a step of inclusion in paraffin and / or of production of histological sections.

7. A method according to any preceding claim, wherein the quantification of the immunolabeling is carried out after acquisition of images by fluorescence photon microscopy.

8. A method according to any preceding claim, wherein a nuclear counterstaining step is carried out using a nuclear dye, for example Hoechst dye.

9. Use of a method as defined in any one of claims 1 to 8, for evaluating the effect of a product or an active ingredient on epidermal cell regeneration.

10. Use according to claim 9, wherein the effect is an effect on the skin, in particular of the face, for example on the signs of aging, maintaining or improving the barrier function of the skin, in particular of the skin of the face.

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