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) Culturing the epidermal fragment on a support, 2) Detaching the epidermal fragment from the support, 3) Performing immunostaining 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 immunostained nuclei of the proliferative cells by image processing, 7) Quantifying cell division by counting the nuclei through image analysis. The present invention also relates to using the method to evaluate the effect of a product or 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 its use to evaluate the effect of a product or an active ingredient on epidermal cell regeneration.

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

[0003] The skin is a vital organ in its own right, composed of three distinct tissues, each assuming different roles through different cell types and structures.

[0004] The outermost tissue, and therefore the most exposed to external stimuli, is the epidermis. This stratified keratinized epithelium (Malpighian) is composed of different cells associated with numerous barrier and protective functions. The predominant cells are keratinocytes, which divide in the basal layer and begin their differentiation up to the stratum corneum (the outermost layer), then are shed by desquamation, on average in 21 to 28 days. 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-related, and bacteriological aggressions. This protection is ensured in particular by the most differentiated layers and the stratum corneum, known for its hydrophobic properties and its compact and impermeable appearance.Proper differentiation, and therefore proper desquamation, is essential for a smooth, homogeneous, and even skin surface. All of these mechanisms depend directly on the proliferative capacity of keratinocytes. However, external stressors, such as UV radiation, and age damage keratinocytes and slow down this process, which can then become longer and more irregular. As desquamation becomes irregular and / or chaotic, imperfections in the smoothness and homogeneity of the skin surface develop.

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

[0006] Before marketing any cosmetic or dermatological product, it is necessary to test its efficacy, particularly on epidermal cell renewal. For this purpose, it is common practice to use in vitro skin models, notably reconstructed human epidermis (also called "HRE" for Reconstructed Human Epidermis) or skin equivalents. However, the histological techniques classically used consist primarily of performing cross-sections of RHE, which only provide partial information.

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

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

[0009] Description of the invention

[0010] The present invention is specifically designed to address these needs and drawbacks of the prior art.

[0011] The inventors have indeed developed a technique allowing the surface of a skin model to be marked and observed as a whole.

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

[0013] The inventors of the present invention have indeed developed a method enabling, on the one hand, the measurement of cell proliferation over the entire surface of the basal layer of the epidermal model, and on the other hand, the obtaining of cell proliferation measurements over the entire epidermal model. Advantageously, the results obtained do not result from extrapolation, as is generally the case in prior art methods, but are indeed the direct result of implementing 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, particularly 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 the in vitro characterization and quantification of proliferative cells in a fragment of epidermis, comprising the following steps:

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

[0017] 2) Detach the epidermal fragment from the substrate,

[0018] 3) Perform immunostaining on the epidermal fragment,

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

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

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

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

[0023] A second object 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 this invention, "characterizing proliferative cells" means visualizing and identifying, using immunostaining, a proliferation marker present in keratinocytes and expressed only during the division phases of the cell cycle. Characterizing proliferative cells means identifying those that express this marker. The proliferation marker can be any marker known to those skilled in the art. For example, it could 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 could be Ki-67.

[0025] For the purposes of this invention, an "epidermal fragment" means any tissue structure exhibiting the same structural characteristics, particularly three-dimensional characteristics, as those of the superficial layer of skin covering the dermis. As such, the epidermal fragment also includes a basal layer. Advantageously, the epidermal fragment reproduces the behavior of the epidermal layer of the skin. It could be, for example, a reconstructed human epidermis or a skin graft.

[0026] For the purposes of this invention, a "proliferative cell" is defined as any healthy cell that reproduces by mitosis, that is, by dividing itself, into a line of healthy cells. A qualitative and / or quantitative level of proliferative cells in the epidermal fragment that is higher than a reference level of proliferative cells in healthy epidermis indicates improved cell renewal. Conversely, a qualitative and / or quantitative level of proliferative cells in the epidermal fragment that is lower than a reference level of proliferative cells in healthy epidermis indicates degraded cell renewal. The reference level may be the level measured in a healthy epidermis, or the control level measured on the epidermal fragment before any treatment thereof.

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

[0028] The support for the epidermal fragment can be any support known to those skilled in the art for this use. For example, it could be a polycarbonate membrane or a collagen matrix. The support is in contact with the basal layer of the epidermal fragment.

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

[0030] Following the decoupling step, the epidermal fragment can be preserved in such a way as to prevent any degradation by a method known to those skilled in the art. For example, the epidermal fragment can be fixed in paraformaldehyde.

[0031] Step 3) of immunolabeling the epidermal fragment can be carried out using any method known to those skilled in the art. This could be, for example, direct immunolabeling with 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 can be coupled to a fluorophore, for example Alexa 488. Otherwise, a secondary antibody coupled to a fluorophore must be used to highlight the selected marker. Advantageously, immunolabeling contributes, in subsequent steps of the process, to the visualization and / or selection and / or quantification of proliferating cells.

[0032] Optionally, a permeabilization step of the detached epidermal fragment can be performed before immunostaining, in order to allow the antibodies to penetrate the cell nucleus. Advantageously, this allows for more effective analysis. given that the keratinocyte cell renewal process involves nuclear markers.

[0033] Optionally, a nuclear counterstaining step can be performed using a nuclear dye, for example, Hoechst stain. This step allows visualization of the proportion of proliferative cells among all the cells present in the basal layer of the epidermis. It also makes it possible to calculate a proliferative cell / total cell ratio, allowing for a more precise analysis and comparison of the different conditions tested.

[0034] Step 4) of acquiring images of the basal layer of the epidermal fragment over its entire surface can be carried out by any suitable means known to those skilled in the art, for example, by means of a fluorescence light microscope with a 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 microscopic resolution.

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

[0036] Step 7) of quantifying cell division by counting immunolabeled nuclei by image analysis can be done by any means commonly used for this purpose by a person 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 paraffin embedding step and / or the preparation of histological sections. This is an advantage compared to most prior art methods, which cannot dispense with these steps.

[0038] Advantageously, it is possible to treat the epidermal fragment, during step 1) of its culture, with a product or active ingredient, for example, a cosmetic or dermatological one, to test the effect of the product or active ingredient on epidermal cell proliferation and / or regeneration in the epidermal fragment by implementing the process of the invention. In this case, it is possible to compare the effect of the product or active ingredient to a control, for example, a positive control prepared with any substance known to induce 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 a healthy epidermis.

[0039] Any product or active ingredient may 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 also be used to evaluate the advantageous effect of a product or active ingredient on epidermal cell regeneration, particularly on the skin, especially the face, for example, on the signs of aging, or on maintaining or improving the skin's barrier function. For example, the effect on the signs of aging may be a resurfacing, smoothing effect, particularly on wrinkles and fine lines, and / or a plumping effect.

[0041] Other advantages may become apparent to a person skilled in the art upon reading the examples below, illustrated by the accompanying figures, which are given by way of illustration. Brief description of the drawings

[0042] Fig. 1 shows photos of untreated models, photos of models treated with KGF and photos of models treated with anti-aging care product 2 or anti-aging care product 3 (visualization after immunolabeling, proliferating cells labeled positively by Ki-67 appear in white).

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

[0044] Examples

[0045] Example 1: Evaluation of anti-aging skincare formula 1 on keratinocyte renewal 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 reproduces the characteristics and behavior of the epidermal layer of the skin, thus allowing the evaluation of the effect of an active ingredient or formula on the ability of keratinocytes to proliferate. This model allows us to observe the cell renewal of basal keratinocytes, which are central to epidermal renewal, and thus demonstrate a beneficial effect of the anti-aging skincare formula 1. These reconstructed epidermis also have the advantage of being reproducible because they are derived from characterized cell 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 they 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 is in optimal conditions for conducting the experiment.

[0049] Treatment of reconstructed epidermis:

[0050] The RHEs are treated or not (control) the following day (D0) by topical application of the anti-aging care formula 1 at a dose of 2 mg / cm2, representing 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] Treatment was stopped after 5 days of incubation. The protocol followed to detach the basement membrane from its support and for immunostaining is as follows: - Application of the Dispase II® solution (2.4 IU / mL, Sigma) directly into the well for 2 min at room temperature - Detaching the base layer of the polycarbonate membrane using pliers - Fixation with 2% paraformaldehyde for 1 hour - Rinse with PBS for 5 minutes x 2 - Permeabilization of the RHE with a Triton solution - Saturation of non-specific antigenic sites with a BSA solution - Specific labeling with an antibody directed against the Ki-67 protein and coupled to the Alexa 488 fluorophore (Abcam) - Hoechst nuclear counterstaining (nuclear labeling)

[0054] Microscopic characterization:

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

[0056] The parameters are shown in Table 1.

[0057] [Tables 1] Dye / marking 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 mosaic images was performed using a macro (an automated sequence of commands enabling image processing and analysis) on the FIJI image analysis software. Using this macro, 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 that there is a significant increase in the number of proliferating nuclei after treatment with the tested product.

[0061] Results:

[0062] The untreated samples show homogeneity in preparation and labeling. The average quantity of nuclei renewed across the 8 models is 21225 nuclei + / - 28%.

[0063] Samples treated with the positive control KFG are homogeneous and appear visually whiter (therefore more stained by KI67) than untreated samples. The number of renewing nuclei increases significantly compared to untreated models, with an average of 33,875 nuclei + / - 32%.

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

[0065] Conclusion: The specific method developed makes it possible to quantify dividing cells across the entire surface of the reconstructed epidermis. Furthermore, the anti-aging skincare formula 1 induces significant cell proliferation after 5 days of treatment compared to untreated RHE models.

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

[0067] The reconstructed human epidermis (“HRE”, Episkin™ RHE / S / 10) is a skin model that reproduces the characteristics and behavior of the epidermal layer of the skin, thus allowing the evaluation of the effect of an active ingredient or formula on the ability 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 demonstrate a beneficial effect of the tested treatment. 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 they 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 is in optimal conditions for conducting the experiment.

[0070] Treatment of reconstructed epidermis:

[0071] The RHEs are treated or not (control) the following day (D0) by topical application of the two anti-aging treatments at a dose 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 skincare 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] Treatment was stopped after 5 days of incubation. The protocol followed to detach the basement membrane from its support and for immunostaining is as follows: - Application of the Dispase II® solution (2.4 IU / mL, Sigma) directly into the well for 2 min at room temperature - Detaching the base layer of the polycarbonate membrane using pliers - Fixation with 2% paraformaldehyde for 1 hour - Rinse with PBS for 5 minutes x 2 - Permeabilization of the RHE with a Triton solution - Saturation of non-specific antigenic sites with a BSA solution - Specific labeling with an antibody directed against the Ki-67 protein and coupled to the Alexa 488 fluorophore (Abcam) - Hoechst nuclear counterstaining (nuclear labeling)

[0075] Microscopic characterization:

[0076] The models were characterized by epifluorescence (Leica DM6) with the xlO objective in fluorescence - Leica DAP filters for highlighting Hoechst labeling and with L5 filter for Ki-67 labeling (see [Fig.1]).

[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 mosaic images was performed 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 that there is a significant increase in the number of proliferating nuclei after treatment with the tested product.

[0082] Results:

[0083] The untreated samples show homogeneity in preparation and labeling. The average quantity of nuclei renewed across the 6 models is 14911 nuclei + / - 17%.

[0084] Samples treated with the positive control KFG are homogeneous and appear visually whiter than untreated samples. The number of renewing nuclei increases significantly compared to untreated models, with an average of 55,755 nuclei + / - 10%.

[0085] Samples treated with the anti-aging care formula 2 are homogeneous in terms of labeling. The number of renewing nuclei increases very slightly compared to 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 labeling. The number of renewing nuclei 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. Thus, the anti-aging skincare formula 3 induces significant cell proliferation after 5 days of treatment compared to untreated RHE models.

Claims

Demands

1. Method for the in vitro characterization and quantification of proliferative cells in an epidermal fragment, comprising the following steps: 1) Culturing the epidermal fragment on a support, 2) Detaching the epidermal fragment from the support, 3) Performing 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 proliferating cells by image processing, 7) Quantifying cell division by counting the nuclei by image analysis.

2. Method according to claim 1, wherein said epidermal 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 epidermal fragment.

3. Method according to claim 1 or 2, wherein the epidermal fragment is a reconstructed epidermis or a skin graft.

4. Method according to any one of the preceding claims, wherein said support is selected from a polycarbonate membrane or a collagen matrix.

5. A method according to any one of the preceding claims, wherein the immunolabeling is a direct immunolabeling by antibodies directed against at least one cell proliferation marker, for example selected 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 paraffin embedding step and / or the preparation of histological sections.

7. A method according to any one of the preceding claims, wherein the quantification of immunostaining is carried out after image acquisition by fluorescence photon microscopy.

8. A method according to any one of the preceding claims, 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, to evaluate the effect of a product or active ingredient on epidermal cell regeneration.

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

Images