Method for evaluating the impact of a stimulus and / or treatment on the growth and / or condition of a hair

Abstract

Method for evaluating the impact of a stimulus and / or treatment on the growth and / or condition of a hair Method for evaluating ex vivo the effect of a stimulus and / or treatment, affecting at least the mechanical and / or geometric properties of the connective sheath of a hair follicle (1) of a human hair, on the growth and / or condition thereof, the method comprising the steps of: a) acquiring with an imager (2, 12, 28) during an observation period at least one image of the hair follicle having been exposed to the action of the stimulus and / or treatment, the hair follicle being arranged in a culture medium (5), b) generating at least one item of information relating to the growth and / or condition of said hair, from the acquired image(s), c) comparing the item(s) of information generated in step b) with at least one reference datum, and delivering to the less information relating to the action of the stimulus and / or treatment on the growth and / or condition of the hair.Figure : 13.

Description

Title of the invention: Method for evaluating the impact of a stimulus and / or treatment on the growth and / or condition of a hair Technical field

[0001] The present invention relates to methods for evaluating the growth dynamics of a human hair and / or highlighting the effectiveness of at least one product on the growth and / or condition of this hair. Prior art

[0002] The hair follicle, also called pilosebaceous follicle, is a structure anchored in the dermis of the skin giving rise to the hair shaft. This follicle is made up of several concentric internal layers of cells, surrounded successively by an external epithelial sheath and a connective tissue sheath. The connective tissue sheath, also called connective sheath or dermal sheath, provides a support function for the hair. It is composed of longitudinal layers of collagen fibers and also contains blood vessels and nerve endings. At the base of the hair follicle is the bulb, the "living" part of the hair where cell division occurs. The hair follicle has the property of regenerating cyclically.This process follows three phases: an active growth phase called the anagen phase, an involution phase or catagen phase, and a resting phase with hair loss, called the telogen phase. Following this last phase, the follicle regenerates through a process of neo-morphogenesis, notably from a reservoir of pluripotent stem cells located in a bulge in the middle of the hair follicle.

[0003] The study of the hair follicle is thus valuable for understanding the mechanisms underlying the growth of hair and hair, and their impact on the apparent state of the latter, in particular on their thickness.

[0004] Many studies have focused on hair follicles, particularly the movements of the different types of cells within them. However, most of the experiments and observations on which current scientific knowledge is based have been carried out on animals, particularly rodents. Human hair follicles, however, have important differences from animal hair follicles, particularly with regard to their hormonal regulation.

[0005] A few rare experiments on human hair have been recorded but they are based exclusively on comparisons of photos taken at relatively long time intervals on samples fixed or frozen in such a way that they do not allow tracking of the movements of an individual cell in the hair follicle during growth.

[0006] There thus remains a need to obtain more information on human hair than current methods allow, for example to facilitate the development of new hair cosmetic compositions. Summary of the invention

[0007] The invention aims to meet this need and has as its subject, according to one of its aspects, a method for evaluating ex vivo a growth dynamics of at least one human hair or hair, having a hair follicle, the method comprising the steps consisting of: a. imaging the hair follicle, placed in a culture medium, with an imager during a sufficiently long observation period with a sufficiently fast acquisition frequency, and a sufficiently fine resolution where appropriate, to obtain at least one animated sequence of the growth of the hair or hair in this culture medium, b. generate from this sequence at least one piece of information relating to the dynamics of hair growth.

[0008] The animated sequence can make it possible to observe movements of cells constituting the hair follicle with certain types of imager having a sufficiently fine resolution, such as for example with a multiphoton microscope. Other types of imager can make it possible to observe, such as the bright field microscope, groups of cells indistinctly. Other types of imager can only make it possible to observe the dynamics of hair growth, such as for example the holographic microscope.

[0009] The method according to the invention makes it possible to acquire an animated sequence of the hair follicle in the active growth phase by imaging it in a culture medium which preserves and ensures its growth. Obtaining such an animated sequence makes it possible to draw more precise observations than a comparison between two images separated by a relatively large time interval of frozen samples for example. Indeed, an animated sequence makes it possible to first of all decide on the linearity of the growth. It also makes it possible to precisely follow the movement of cells within the hair follicle during growth. It is also possible to observe the cell divisions of the progenitor epithelial cells, located at the base of the hair follicle, ensuring the production of the hair. Animated sequence

[0010] By "animated sequence" is meant a set of images sufficiently close in time to the observed phenomenon to enable the trajectory of the hair follicle cells to be followed.

[0011] Image acquisition is preferably carried out at a frequency of between 2 images per hour and 30 images per hour. This image acquisition frequency is fast enough to track cell movements and observe cell divisions within the hair follicle, thereby providing more precise information on cell behavior. Image acquisition can last for at least 24 hours and up to 7 days depending on the type of imager used.

[0012] The sequence may comprise at least 48 images, better still at least 100 images, even better still at least 1000 images, in particular more than 5000 images.

[0013] By "sufficiently high resolution" is meant a resolution allowing two points separated by a sufficiently small distance to be distinguished to allow the movement of cells within the hair follicle to be observed.

[0014] The resolution of the imager may be less than or equal to 250 nm in xy.

[0015] The display of the images can be done in accelerated mode, in particular at a frequency of at least 10 images per second. Between two images of the sequence, a maximum displacement of a cell can represent on the image less than 1 / 10 of the diagonal of the image.

[0016] The images in the sequence may be monochrome or color, depending on the type of imager.

[0017] The animated sequence may display a date and / or a scale.

[0018] The animated sequence may be generated in the form of a video having a frequency of predefined images or of a sequence of images whose transition from one image to another is controlled by a user, or of a set of images displayed side by side, or of a projection in a single image of several images, in particular of all the images of the animated sequence.

[0019] The sequence may include all the acquired images or only a selection of them, for example one image out of n. Information

[0020] “Growth dynamics information” may include a measurement of the growth rate, information on the linearity of the growth, information on the behavior and / or movement, in particular on the speed and / or direction of movement, of at least one cell of the hair follicle during growth, a quantification of the number of cell divisions in a region of the hair follicle, information on the direction of cell divisions during growth within the different layers of the hair follicle, and / or information on an evolution of a characteristic of the hair follicle during growth.

[0021] The method may include a step c) where said information is compared with at least one reference datum. This is, for example, data obtained for hairs or hairs from the same person, but subjected to a different environment during growth. Imager

[0022] The hair follicle can be imaged in 2D or 3D over time.

[0023] In one embodiment, the imager is a fluorescence microscope, particularly a multiphoton microscope. These microscopes can image the hair follicle in 3D by building up a stack of images by varying the depth of the imaging plane within the hair follicle.

[0024] In this embodiment, at least one hair follicle can be received in a sample holder comprising a support defining at least one cavity intended to be filled with the culture medium, comprising a bottom and having at least one portion raised relative to the bottom and below the maximum filling level of the cavity, this portion having at least one hole of dimensions adapted to hold a human hair or hair substantially vertically, and at least one groove arranged so as to hold a human hair or hair substantially horizontally. Said portion can be in the form of a pad. The latter can comprise two uprights supporting an upper surface, extending substantially horizontally, the well(s) and the groove(s) of the sample holder being arranged within this surface.

[0025] At least one hair follicle may be disposed in a hole of the sample holder and at least one other hair follicle may be disposed in a groove of the sample holder.

[0026] By placing the hair follicles in this way, it is possible to acquire with the fluorescence microscope, in particular multiphoton, respectively a longitudinal sectional image of the hair follicle, the longitudinal axis of the follicle being the axis of the hair shaft, or a cross-sectional image, perpendicular to the axis of the hair shaft. By imaging the hair follicles in the direction of the axis of their shaft and perpendicular to this axis, a better characterization of the hair follicle is obtained by fluorescence microscopy, in particular multiphoton.

[0027] The sample holder may be closed by a transparent structure, for example a glass coverslip.

[0028] In this embodiment, at least one constituent of the hair follicle can be marked using an imaging marker. Several constituents of the hair follicle can be marked using the same imaging marker or different imaging markers. At least one piece of information relating to the movements of said marked constituent during the development of the hair can be generated from the acquired animated sequence.

[0029] By "constituent of the hair follicle" we mean both molecules, such as DNA or proteins, for example actin, but also by extension and misuse of language, components of the hair follicle such as fibers, cells, or cellular structures such as nuclei.

[0030] Labeling the constituents of the hair follicle makes it easier to track their movements in the animated sequence, during the hair growth process, with a view to deducing information to characterize the dynamics of hair growth. It is also possible to rely on the natural autofluorescence of certain constituents of the hair follicle, such as that of a coenzyme, nicotinamide adenine dinucleotide (NADH), present in living cells.

[0031] At least one piece of information relating to the movements of cells during hair growth can be generated based on their natural autofluorescence, in particular due to the presence of nicotinamide adenine dinucleotide (NADH).

[0032] The hair follicle comprises eukaryotic cells, and in one embodiment, the nuclei of these cells can be labeled using an imaging marker, in particular using a dye, for example a Hoechst dye, in particular Hoechst-33258. At least one piece of information relating to the movements of the cells with the labeled nucleus during hair growth can be generated from the analysis of the acquired animated sequence.

[0033] At least one piece of information relating to the divisions of cells with a marked nucleus during the development of the hair can be generated from the analysis of the animated sequence.

[0034] Hoechst dyes are used to label DNA, and thus by extension allow the labeling of nuclei containing DNA in eukaryotic cells. Labeling nuclei makes it easier to follow the movement of cells during hair growth, to analyze their movement, to calculate their speed within the different layers forming the hair follicle but also to estimate the number of cell divisions.

[0035] In another embodiment, the imager is a brightfield microscope.

[0036] The resolution of the bright field microscope can be about 250 nm in xy and about 1 pm in z.

[0037] In another embodiment, the imager is a holographic microscope

[0038] The resolution of the holographic microscope may be about Ipm in xy.

[0039] The use of a holographic microscope makes it possible to image the hair by leaving it in an incubation medium, without having to place it in a specific sample holder closed in particular by a coverslip. Observation period

[0040] The observation period can make it possible to observe a growth of at least 0.1 mm, in particular between 0.1 and 2.5 mm.

[0041] The observation period may be greater than or equal to 12 hours, better still 24 hours.

[0042] In an embodiment where the imager is a multiphoton microscope, the observation period may be between 1 hour and 48 hours, preferably at least 24 hours.

[0043] In an embodiment where the imager is a bright field microscope or a holographic microscope, the observation period may be greater than or equal to 48 hours.

[0044] In an embodiment where the imager is a bright field microscope, the observation period may be between 1 hour and 120 hours, preferably at least 48 hours.

[0045] In an embodiment where the imager is a holographic microscope, the observation period may be between 1 hour and 170 hours, preferably at least 72 hours.

[0046] Bright field microscopy and holographic microscopy are less phototoxic than fluorescence microscopy, especially multiphoton, which allows the hair follicle to be kept in the growth phase for longer. Hair follicle

[0047] The hair follicle can be extracted in the anagen phase.

[0048] The hair follicle may be extracted from human tissue. The human tissue may be a Tissue scraps resulting from surgery, particularly cosmetic surgery, such as a facelift. Follicles can still be extracted using the same techniques as for hair transplants.

[0049] Hair follicles can still be grown in a laboratory.

[0050] The follicle can be extracted under a stereomicroscope. It can be extracted by means of a scalpel and / or tweezers.

[0051] The hair follicle may be placed in a culture medium. The culture medium may comprise William's E medium. The medium may be enriched with glutamine and / or hydrocortisone and / or insulin. The culture medium may be the medium described in the article "Human hair growth in vitro" by Philpott et al., Journal of Cell Science, 1990.

[0052] The hair follicle can be incubated in the culture medium under the temperature, humidity and CO2 conditions described in the article “Human hair growth in vitro” by Philpott et al cited above, before being imaged.

[0053] The hair follicle may be a non-pigmented follicle. Choosing a non-pigmented follicle prevents melanin, present in pigmented follicles, from absorbing the light used for excitation when the imager is a fluorescence microscope, resulting in greater phototoxicity for the follicle and sometimes saturation of the fluorescent signal emitted by the melanins.

[0054] The hair follicle can be cut above its bulb before being placed in the culture medium and then imaged. After being extracted and before being cut, the hair follicle can be placed in the culture medium. The hair follicle can be incubated in the culture medium after cutting and before being imaged.

[0055] The method may thus comprise the step of cutting the hair follicle above the bulb using a scalpel before placing it in the culture medium and then imaging it.

[0056] Methods for evaluating the effect of a stimulus and / or treatment on the growth dynamics of a hair or a lock of hair

[0057] According to another of its aspects, the invention relates to a method for ex vivo evaluating the effect of a stimulus and / or a treatment on the growth dynamics of at least one human hair or lock of hair, comprising the steps of: a. assess the dynamics of hair growth using the method as previously defined, in particular during an observation period of between 1 and 24 hours, b. repeat step a) on at least one hair exposed to the action of the stimulus and / or treatment, c. compare the information generated by the two assessments and generate at least one piece of information relating to the action of the stimulus and / or treatment on the dynamics of hair growth.

[0058] The hair or hair exposed in step b) to the action of the stimulus and / or treatment may be the same hair or hair as the hair or hair of step a). Alternatively, the hair or hair exposed in step b) to the action of the stimulus and / or treatment may be another hair or hair than the hair or hair of step a), preferably coming from the same person.

[0059] Step b) can repeat step a) at any point on the hair(s) exposed to the action of the stimulus and / or treatment using an observation period of the same duration.

[0060] The exposure to the action of the stimulus and / or treatment may be brief or continuous in time, for example the exposure may extend over the entire duration of step b).

[0061] In an embodiment where the impact of a treatment, in particular of an active ingredient, is evaluated, the hair may be exposed to the treatment throughout the observation period necessary for the evaluation of its growth dynamics.

[0062] The invention also relates to a method for evaluating ex vivo the effect of a stimulus and / or a treatment on the growth dynamics of at least one human hair, in which at least two populations of at least one human hair are exposed differently to the stimulus and / or to the treatment, in which at least one piece of information relating to the growth dynamics of the hair, obtained by means of the method for evaluating the growth dynamics as previously defined, is compared for the different populations.

[0063] Exposure to the stimulus may be different in duration between populations. Treatment may be applied at different doses between populations.

[0064] One of the populations may not be exposed to the stimulus and / or treatment, unlike the other population(s). Stimulus and treatment

[0065] The stimulus according to the invention can be chosen from an electrical, chemical, biological, mechanical, thermal, electromagnetic, in particular luminous, or radiological stimulus, or a combination of these.

[0066] The treatment may be non-therapeutic. The treatment may be a cosmetic treatment, in particular via the application of a product promoting the regrowth or beautification of the hair, in particular the thickening of the hair.

[0067] The treatment may include the application or administration of a product, in particular a cosmetic product.

[0068] By “cosmetic product” is meant a product as defined in Directive 93 / 35 / EEC of June 14, 1993, amending Directive 76 / 768 / EEC. System

[0069] According to another of its aspects, the invention relates to a system for implementing a method as previously defined, comprising an imaging unit, in particular a fluorescence microscopy unit, in particular multiphoton or a bright field microscopy unit or a holographic microscopy unit, making it possible to acquire at least one animated sequence of at least one human hair, placed in a culture medium, and a calculation means, in particular a computer unit capable of storing the animated sequence(s) and / or processing them by means of an integrated processor or sending them to a remote calculation server, this calculation means being configured to generate from the acquired animated sequence(s), at least one item of information relating to the dynamics of hair growth, and to present this information, for example in the form of text and / or images, for example via a user interface.

[0070] The imaging unit may be separated from the rest of the system and connected to it, for example, by a wired or wireless connection.

[0071] The computing means may comprise a processor integrated into the system or be remote, such as a computing server connected to the system by a wired or wireless connection.

[0072] The user interface may be included in the system or separate, such as when it is a display screen. Sample holder

[0073] According to another of its aspects, the invention also relates to a sample holder comprising a support defining at least one cavity intended to be filled with a liquid culture or preservation medium, comprising a bottom and having at least one portion raised relative to the bottom and below the maximum filling level of the cavity, this portion having at least one hole of dimensions adapted to hold a human hair or hair substantially vertically, and at least one groove arranged so as to hold a human hair or hair substantially horizontally. Said portion may be in the form of a pad. The latter may comprise two uprights supporting an upper surface, extending substantially horizontally, the well(s) and the groove(s) of the sample holder being arranged within this surface.

[0074] The sample holder according to the invention provides a support making it possible to image hairs or hairs in the direction of the axis of their stem but also perpendicular to this axis, which is particularly advantageous when the hairs or hairs are imaged by a fluorescence microscope, in particular a multiphoton microscope as described previously.

[0075] The sample holder allows the hairs or hair to be held in two directions to be imaged while allowing them to grow due to the presence of a culture medium in which the hairs or hair are immersed.

[0076] The sample holder may be at least partially made of a transparent or translucent material.

[0077] The sample holder can be 3D printed, in particular using a photopolymer resin.

[0078] Methods for evaluating the effect of a stimulus and / or a treatment on the growth and / or condition of a hair

[0079] According to another of its aspects, the invention relates to a method for evaluating ex vivo the effect of a stimulus and / or a treatment, affecting at least the mechanical and / or geometric properties of the connective sheath of a hair follicle of a human hair, on the growth and / or the state thereof, the method comprising the steps consisting of: a. acquire with an imager during an observation period at least one image of the hair follicle having been exposed to the action of the stimulus and / or treatment, the hair follicle being placed in a culture medium, b. generate at least one piece of information relating to the growth and / or condition of said hair, for example its thickness, from the image(s) acquired, c. compare the information generated in step b) with at least one reference datum, for example a reference thickness, and deliver at least one piece of information relating to the action of the stimulus and / or treatment on the growth and / or condition of the hair.

[0080] By "the mechanical and / or geometric properties of the connective tissue sheath" is meant at least one of the characteristics of the connective tissue sheath chosen from the following non-limiting list: the rigidity of the connective tissue sheath, its local or global integrity, its fibrillar organization and its thickness.

[0081] The reference data may be acquired on at least one hair or a reference population, not exposed to said stimulus and / or treatment, and / or exposed differently to said stimulus and / or treatment.

[0082] The method may comprise the step of exposing the hair follicle to said stimulus and / or treatment before placing it in the culture medium and imaging it.

[0083] According to another of its aspects, the invention also relates to a method for evaluating ex vivo the effect of a stimulus and / or a treatment affecting at least the mechanical and / or geometric properties of the connective sheath of a hair follicle of a human hair on the growth and / or condition thereof, in which at least two populations of at least one human hair are exposed differently to the stimulus and / or to the treatment, in which at least one piece of information relating to the growth and / or to the condition of the hair generated from at least one image acquired with an imager during an observation period of each population placed in a culture medium is compared between the populations.

[0084] As previously indicated, exposure to the stimulus may be different in duration between populations. The treatment may be applied at different doses between populations. One of the populations may not be exposed to the stimulus and / or treatment, unlike the other population(s).

[0085] The stimulus may be chosen from a mechanical, electrical, chemical, biological, thermal, electromagnetic, in particular luminous, or radiological stimulus, or a combination of these.

[0086] The stimulus may be a mechanical stimulus and the stimulus may comprise severing the connective tissue sheath over at least a portion of its circumference, for example over its entire circumference. The severing may be performed above the bulb of the hair follicle, preferably less than one millimeter above the bulb. The severing may be performed using a scalpel.

[0087] The stimulus may include severing the hair follicle above the bulb using a scalpel.

[0088] The stimulus may be a mechanical stimulus and the stimulus may comprise the punctate alteration, at one or more points, of the connective sheath. This alteration may be carried out above the bulb of the hair follicle. It may be carried out by photoablation.

[0089] The stimulus may include exposure to an enzyme, particularly an enzyme having an action on collagen fibers such as collagenase.

[0090] The method may comprise the step of exposing the hair follicle to said stimulus and / or treatment before placing it in the culture medium and imaging it.

[0091] The information relating to the growth and / or the state of said hair or hair may concern at least one of the characteristics chosen from its thickness, its porosity and / or its hydration, the organization of its scales, the organization of its keratin fibers, its growth speed, the number of cell divisions in its bulb, its rigidity, its shape and its direction of growth after exposure to the stimulus and / or treatment.

[0092] The imager may be an imager as previously defined and may have any of the characteristics as defined above.

[0093] The hair follicle may exhibit any of the characteristics as previously defined.

[0094] The treatment may have any of the characteristics as previously defined.

[0095] According to another of its aspects, the invention also relates to a method for screening an active ingredient in which the effect of various active ingredients on the growth and / or condition of at least one human hair or hair is evaluated ex vivo by means of a method for evaluating the impact of a stimulus and / or treatment as previously defined, so as to identify at least one active ingredient having a desired action on the hair or hair.

[0096] By "desired action" is meant any action improving a characteristic of the hair, such as the organization of its scales, the organization of its keratin fibers, its growth speed, the number of cell divisions in its bulb, its thickness and its porosity or hydration. Brief description of the drawings

[0097] The invention may be better understood by reading the description which follows, non-limiting examples of its implementation, and by examining the attached drawing, in which:

[0098] [Fig.l] represents in a schematic view the different stages of an example of a method for evaluating the growth dynamics of a hair or hair in accordance with the invention,

[0099] [Fig.2] illustrates a bright field microscope imaging a hair follicle of a hair, according to the method of [Fig.l],

[0100] [Fig.3] is a diagram of an animated sequence acquired by an imager, in particular the imager of [Fig.2], according to the method of [Fig.l],

[0101] [Fig.4] represents two images extracted from an animated sequence acquired during the process represented in [Fig.l] with the imager of [Fig.2]

[0102] [Fig.5] illustrates a multiphoton microscope imaging a hair follicle of a hair in two different positions, according to the method of [Fig.l],

[0103] [Fig.6] is a schematic and partial perspective view of a sample holder according to the invention,

[0104] [Fig.7] represents two images extracted from an animated sequence acquired during the process represented in [Fig.l] with the imager of [Fig.5], the hair follicle being imaged longitudinally in the direction of the hair shaft

[0105] [Fig.8] presents in a) an image extracted from an animated sequence acquired during the process represented in [Fig.l] with the imager of [Fig.5], the hair follicle being imaged transversely, perpendicular to the direction of the hair shaft, and in b) a projection of the images of the entire sequence making it possible to highlight the trajectory of the 3 cells followed for a certain time,

[0106] [Fig.9] represents an image of the density of mitoses within a hair follicle, calculated from the animated sequence of [Fig.7],

[0107] [Fig. 10] illustrates a holographic microscope imaging several hair follicles, according to the method of [Fig.l],

[0108] [Fig. 11] represents an image extracted from an animated sequence acquired during the process represented in [Fig.l] with the imager of [Fig.10],

[0109] [Fig. 12] is a schematic and partial representation of step a0) of the method of [Fig.l],

[0110] [Fig. 13] shows an image acquired during a process evaluating the impact of a stimulus and / or treatment affecting the mechanical and / or geometric properties of the connective sheath of a hair follicle of a hair on the growth and / or condition of at least one hair,

[0111] [Fig. 14] shows images of a reference population and a stimulus-affected population acquired in a process similar to that of [Fig.13], and

[0112] [Fig. 15] illustrates an example of a system for implementing a method for evaluating the growth dynamics of a hair or hair according to the invention, Detailed description

[0113] We will describe with reference to figures 1 to 12 different steps of an example of a method for ex vivo evaluation of the dynamics of hair growth.

[0114] Step a) of [Fig. 1] may consist of imaging one or more hair follicles of hairs or hairs arranged in a culture medium, such as William's E medium enriched with glutamine, insulin and hydrocortisone with an imager. Step a) extends over a sufficiently long observation period, with a sufficiently fast acquisition frequency, for example 30 images per minute, and a sufficiently fine resolution to acquire an animated sequence of the growth of the hair or hair in which movements of the cells constituting the hair follicle can be observed.

[0115] Step a) may optionally be preceded by a step a0 where the hair follicle 1 is cut using a scalpel before placing it in the culture medium and imaging it.

[0116] Step b) of the method may consist of generating at least one item of information relating to the dynamics of hair growth from the acquired sequence.

[0117] The method may optionally include a step c) where the generated information is compared with at least one reference datum.

[0118] The hair follicle 1 may be imaged during step a) by a bright field microscope whose objective 2 is illustrated in [Fig.2]. The hair follicle 1 is received in a sample holder 3, having a groove 4 in which the follicle 1 is placed. The sample holder 3 may be filled with the culture medium so that the groove 4 is immersed by the medium. The sample holder 3 may be closed by a glass coverslip 6, silicone grease 7 being placed under the coverslip at the point of contact with the edges of the sample holder. A drop of oil 8 may be placed on the coverslip 7 so as to eliminate part of the refraction of the rays.

[0119] Figure 3 schematically and partially represents an animated sequence 9 acquired by an imager during step a) of the method of Figure 1. The animated sequence 9 consists of a succession of images 10 acquired at identical time intervals A t. It makes it possible to follow the trajectory of a cell 11 of the hair follicle and to estimate, for example, its speed of movement. The total observation period of step a) of the method of [Fig.l] with a bright field microscope can be greater than or equal to 48 hours, given that the bright field microscope is not very phototoxic, which makes it possible to follow the growth of the hair for a relatively long time.

[0120] Figures 4 a) and 4 b) are two images 10 extracted from the animated sequence 9 acquired with the bright field microscope 2.

[0121] The hair follicle 1 may be imaged during step a) by a multiphoton microscope 12 as illustrated in [Fig.5]. The hair follicle 1 may be received, when imaged with a fluorescence microscope and in particular with a multiphoton microscope, in a specific sample holder 13, shown in isolation in [Fig.6].

[0122] The sample holder 13 comprises a support defining two cavities 14. Each cavity 14 has a bottom 15 and has a part in the form of a pad 16 raised relative to the bottom 15. The pad 16 has two uprights 17 supporting an upper surface 18 extending horizontally. The sample holder 13 is filled with a culture medium 5 such that the hair follicles received are completely immersed. Each pad 16 is thus arranged such that its upper surface 18 is below the maximum filling level of the cavity 14.

[0123] Each pad 14 has on its upper surface 18 a groove 19, arranged so as to hold a human hair or hair substantially horizontally, and two holes 20 of dimensions adapted to hold a human hair or hair substantially vertically.

[0124] The holes 20 may have a diameter of between 200 and 400 μm, for example approximately 300 μm. The groove 19 may have a length of between 3 and 7 mm, for example approximately 5 mm, a width of between 100 and 300 μm, for example approximately 200 μm and a depth of between 50 and 150 μm, for example approximately 100 μm.

[0125] As described previously, the sample holder 13 can be closed by a strip 6 on which a drop of oil is deposited.

[0126] By placing the hair follicles alternately in a groove 19 or a hole 20, the multiphoton microscope makes it possible to acquire respectively a longitudinal sectional image of the hair follicle, the longitudinal axis of the follicle being the axis of the hair shaft, or a cross-sectional image. By varying the imaging plane 21 along a depth axis Z, the multiphoton microscope acquires 3D images and thus 3D animated sequences of the hair follicle.

[0127] The sample holder 13 is for example made of a transparent material. It can be 3D printed using a photopolymer resin.

[0128] Figures 7 a) and 7 b) are two images extracted successively from an animated sequence acquired with the multiphoton microscope 12 in the case where the hair follicle is placed in the furrow 19. Prior to the acquisition of this sequence, the nuclei of the cells were marked with a Hoechst dye to facilitate their visualization.

[0129] Figures 7 a) and 7 b) each show on their left side the image of hair follicle 1 acquired with the multiphoton microscope and on their right side a binary segmentation 22 of this image. The binary segmentation 22 of the image makes it possible to better highlight the cells, which makes them easier to track.

[0130] Four cells in the suprabulbar region of the hair follicle were manually tracked and are indicated in both images. Three cells 23 located in the inner layers of the follicle appear to follow an upward movement in this region while cell 24 indicated in the outer epithelial sheath 25 appears to follow a downward movement.

[0131] [Fig.8] a) is also extracted from an animated sequence acquired with a multiphoton microscope but in a case where the hair follicle is placed in a hole 20 of the sample holder 13. In this example, the nuclei have not been labeled, and the natural autofluorescence of the NADH present in the living cells of the hair follicle is observed. Cells 26 with a strong autofluorescence signal are observed in the outer epithelial sheath 25 in figure a). [Fig.8] b) has been edited to show with arrows the trajectory of the cells 26 between figure a) and figure b). The cells 26 thus appear to follow a rotational movement perpendicular to the hair shaft.

[0132] By combining the information acquired from Figures 7 and 8, it is possible to highlight a complex 3D downward movement, which can be likened to a helix, of the cells of the layers of the external epithelial sheath of hair follicle 1 in its supra-bulbar region.

[0133] Based on the animated sequences, an average speed of movement of the cells can be calculated. Cell 24 located in the outer epithelial layer moves there, for example, with an average speed of - 7 ± 4 pm / h, while the cells in the inner layers move more slowly upwards.

[0134] To characterize the dynamics of hair growth, it is also possible to quantify, from the animated sequence of [Fig.7] for example, the number of cell divisions of the progenitor epithelial cells, located at the base of the hair follicle, during a given period T. By labeling the DNA of the cells with a Hoechst dye, it is possible to observe the equatorial plate on which the chromosomes align during the metaphase of mitosis and thus obtain an estimate of the number of mitoses. [Fig.9] shows a map of the density of mitoses within the hair follicle based on the quantification of the nearest neighbors 27, ranging from 0 to 6, during a period T of approximately 15 hours. This map makes it possible to locate the areas of intense cell division within the follicle.

[0135] The orientation of the equatorial lines forming during the metaphases of mitoses can also be analyzed to estimate the direction of cell divisions and understand the movement of cell flows during hair growth.

[0136] The hair follicle 1 can be imaged during step a) by a holographic microscope 28, as illustrated in [Fig. 10], for example a “Cytonote 6W”, placed in an incubator. Several hair follicles can be observed at a time and are placed in rows of wells 29, filled with culture medium 5.

[0137] [Fig. 11] represents an image extracted from an animated sequence acquired with a holographic microscope 28 during a method according to the invention evaluating the impact of a treatment on the growth dynamics of a hair.

[0138] Three groups of four hair follicles 1 were incubated in wells 29 with a different concentration of hydrocortisone in the culture medium 2.

[0139] The animated sequence has been edited to show a line 30 showing the progression of hair growth under different medium concentration conditions.

[0140] [Fig. 12] represents a partial diagram of step a0) of the method of [Fig. 1] consisting of cutting a hair follicle 1 above its bulb 31 at a cutting line 32 using a scalpel.

[0141] [Fig. 13] shows an image acquired with a bright field microscope during a process evaluating the impact of a stimulus and / or treatment affecting the mechanical and / or geometric properties on the growth and / or condition of at least one hair. Hair follicle 1 was cut using a scalpel as shown in [Fig. 12]. It was then placed in a culture medium and incubated.

[0142] It is observed that in zone 33, corresponding to the regrowth zone after cutting the hair follicle 1 at the cutting line 32, the hair grows back without a connective sheath. The shaft 34 of the hair thickens in zone 33, the thickness Ec measured in the shaft after the cutting line 32 being greater than the thickness E measured in the shaft of the uncut part.

[0143] [Fig. 14] shows images of a reference or control population of hair follicles and a population of hair follicles exposed to the action of a mechanical stimulus, acquired with an imager during a process highlighting the impact of this stimulus on the growth and / or the state of these hair follicles. The stimulus consists of a photoablation of the connective sheath of the hair follicles at eight diametrically opposite points of the sheath, above the bulb of the hair follicles.

[0144] Figures 14 a) and 14 c) represent on day j respectively the control population and the population intended to be exposed to the stimulus just before the photoablation is carried out.

[0145] Figures 14 b) and 14 d) represent respectively the control population and the population exposed to the stimulus on day j + 8.

[0146] The results of the control population can be considered as reference data. By comparing the results of the population exposed to the stimulus with these data, it is shown that photoablation made it possible to relax the constraints mechanical being exposed on the hair shaft, thereby contributing to modifying the shape and direction of hair growth.

[0147] Figure 15 illustrates an example of system D implementing a method for evaluating the hair growth dynamics according to the invention. System D captures, by means of an imaging unit 35, such as a fluorescence microscopy unit, in particular a multiphoton microscopy unit, or a bright-field or holographic microscopy unit, at least one animated sequence of a hair follicle 1, arranged in a culture medium 5. The animated sequence or sequences 9 are stored and processed in a computer unit 36 which sends data for calculation to an integrated 37a or remote 37b processor, communicating with D by any means. The calculation results are returned to the computer unit 36, which transfers at least one item of information in the form of an image and / or text to an interface 38 for a user U.

[0148] The invention is not limited to the examples just described.

[0149] Other imagers may be used and other types of information may be derived from the acquired animated sequences.

[0150] The sample holder may have a different organization, in particular a greater number of grooves and / or wells.

Claims

Claims

1. Method for evaluating ex vivo the effect of a stimulus and / or a treatment, affecting at least the mechanical and / or geometric properties of the connective sheath of a hair follicle (1) of a human hair, on the growth and / or the condition thereof, the method comprising the steps of: a) acquiring with an imager (2, 12, 28) during an observation period at least one image of the hair follicle having been exposed to the action of the stimulus and / or treatment, the hair follicle being arranged in a culture medium (5), b) generating at least one piece of information relating to the growth and / or the condition of said hair, from the acquired image(s), c) comparing the piece(s) of information generated in step b) with at least one reference datum, and delivering at least one piece of information relating to the action of the stimulus and / or the treatment on the growth and / or the condition of the hair.

2. Method according to the preceding claim, the reference data being acquired on at least one hair or a reference population, not exposed to said stimulus and / or treatment, and / or exposed differently to said stimulus and / or treatment.

3. Method according to one of the preceding claims, comprising the step of exposing the hair follicle (1) to said stimulus and / or treatment before placing it in the culture medium (5) and imaging it.

4. Method according to any one of the preceding claims, the information relating to the growth and / or the state of said hair or hair concerning at least one of the characteristics chosen from its thickness, its porosity and / or its hydration, the organization of its scales, the organization of its keratin fibers, its growth speed, the number of cell divisions in its bulb, its rigidity, its shape and its direction of growth after exposure to the stimulus and / or treatment.

5. Method according to any one of the preceding claims, the treatment being a cosmetic treatment, in particular via the application of a product promoting the regrowth or beautification of the hair, in particular the thickening of the hair.

6. A method according to any preceding claim, the imager being a bright field microscope (28).

7. Method according to any one of claims 1 to 5, the imager being a fluorescence microscope, in particular a multiphoton microscope (12), and the hair follicle (1) being received in a sample holder (13) comprising a support defining at least one cavity (14) intended to be filled with the culture medium (5), comprising a bottom (15) and having at least one part (16) raised relative to the bottom and below the maximum filling level of the cavity, this part (16) having at least one hole (20) of dimensions adapted to hold a human hair or hair substantially vertically, and at least one groove (19) arranged so as to hold a human hair or hair substantially horizontally.

8. Method according to any one of the preceding claims, the stimulus being chosen from a mechanical, electrical, chemical, biological, thermal, electromagnetic, in particular luminous, or radiological stimulus, or a combination thereof.

9. Method according to the preceding claim, the stimulus being a mechanical stimulus, the stimulus comprising the sectioning of the connective sheath over at least part of its circumference, in particular above the bulb (31) hair follicle (1), in particular using a scalpel.

10. Method according to claim 8, the stimulus being a mechanical stimulus, the stimulus comprising the punctual alteration, at one or more points, of the connective sheath, in particular carried out by photoablation.

11. Method for screening an active ingredient in which the effect of various active ingredients on the growth and / or condition of at least one human hair or hair is evaluated ex vivo by means of a method according to one of claims 1 to 7, so as to identify at least one active ingredient having a desired action on the hair or hair.

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