Conditioned culture medium derived from human hair follicles and method for producing the same

Abstract

A method for preparing a hair follicle-derived conditioned medium containing one or more bioactive factors, comprising non-invasively harvesting hair follicles from a subject and conditioning the culture medium by culturing the hair follicles in a culture medium for a time sufficient for hair follicle-derived cells to secrete one or more bioactive factors. A hair follicle-derived conditioned medium containing one or more bioactive factors secreted by hair follicle-derived cells non-invasively harvested from a subject. A composition comprising the conditioned medium. Use of the conditioned medium and the composition for therapeutic or cosmetic treatment.

Description

[Technical Field]

[0001] The present specification relates generally to secretome-based therapies, and more specifically to conditioned medium prepared non-invasively using hair follicles. [Background technology]

[0002] Secretome-based therapy has attracted considerable interest in therapeutic and aesthetic fields for the treatment of various conditions, including hair loss, skin disorders, and sports-related injuries. The effectiveness of this therapy is due to the action of various secreted bioactive factors, such as cytokines and growth factors. Collectively, these bioactive factors are referred to as the "secretome." While previous research has provided ample evidence that the use of stem cell-derived secretomes holds great promise as a future therapeutic approach, the need for highly invasive tissue harvesting methods and the limited scalability of the product remain significant limitations that have been overlooked in the prior art.

[0003] In the prior art, secretome therapy has been investigated for various tissues in which stem cells exist. These tissues include bone marrow, adipose tissue, amniotic fluid, and umbilical cord, all of which are harvested using highly invasive methods. An example of a prior art conditioned medium therapeutic currently on the market is bone marrow-derived BabyStem TM , BioCM derived from amniotic fluid TM , and several types of compositions derived from adipose tissue, e.g., AAPE TM , Exomide TM , TheraStem TM , Stem C'rum TMIn addition to the invasive nature of tissue harvesting, these products are allogeneic therapies harvested from continuously expanded cells, with little or no disclosure of information about the cells or donors from which these cells are derived. It is well documented that cells undergo prolonged passage, exhibiting loss or acquisition of various intrinsic properties, including changes in proteomic profiles. It is also now known that the composition of the secretome varies greatly between donors, posing a major challenge for allogeneic therapies. While secretome therapies generally mitigate the safety risks inherent in administering large amounts of live stem cells, the use of allogeneic materials still poses safety concerns.

[0004] Another issue overlooked in the prior art is the inability of secretome therapy to remove residual animal (xenogeneic) serum from the final product formulation. Animal-derived components, such as fetal bovine serum, are widely used in the cultivation of cells and tissues in culture. These animal-derived components raise concerns about reliability and biosafety. While most prior art secretome therapies involve replacing the conditioned medium with a xenogeneic-free medium immediately prior to harvest, issues remain due to the use of animal products at some stage in the cell culture process. A major concern with the use of animal components is the introduction of contaminants during the manufacturing process, which may contaminate the final therapeutic product, rendering it unsuitable for clinical or therapeutic use. Therefore, to ensure clinical safety for human subjects, it is necessary to use xenogeneic-free reagents throughout the entire secretome manufacturing process.

[0005] While the use of serum-free media offers many advantages, such as formulation consistency and biosafety, the potential for suboptimal growth and cellular metabolism leaves an unmet need for alternative methods to enrich secretome compositions that ensure the same level of safety for human use.

[0006] Therefore, there is a need for a novel secretome treatment and secretome preparation method that can overcome the limitations of existing secretome treatments.The present invention discloses a conditioned culture medium that is prepared non-invasively using hair follicles and that may be used for autologous secretome treatment, in which cells are grown using a xeno-free system. Summary of the Invention [Means for solving the problem]

[0007] The present invention relates to a method for non-invasively using hair follicles to produce a conditioned medium containing one or more bioactive factors. Stem cells present in the dermal papilla of hair follicles have been widely studied for their roles in immune regulation and wound healing. Hair follicle stem cells have traditionally been harvested by invasive methods, such as follicular unit extraction (FUE), a technique used for hair transplantation. In various aspects, the present invention is based on the discovery that cells present in the upper region of hair follicles, which can be consistently and non-invasively harvested, can be used to produce secretome therapeutics and are an excellent source of secretome therapeutics. Furthermore, cells present in the upper region of hair follicles can be consistently and non-invasively harvested and easily cultured directly from the hair follicle without the need for digestive enzymes. Anagen-phase human hair follicles are a rich tissue source compared to other stem cell tissue sources and are easily and non-invasively accessible throughout adulthood. The outer root sheath of hair follicles harbors various cell types, including endogenous stem cell populations, responsible for hair growth and development. Therefore, utilizing this tissue source for the production of secretome therapeutics is advantageous.

[0008] According to one embodiment, the preparation method of the present invention comprises: Obtaining hair follicles by non-invasive means; Culturing the hair follicles in a growth culture medium, such as, but not limited to, autologous platelet lysate, for a period of time appropriate for the hair follicle-derived cells to secrete one or more molecules into the growth culture medium, thereby conditioning the growth culture medium; and recovering the resulting conditioned medium for further processing or immediate use. Includes:

[0009] In certain embodiments, the preparation methods of the present invention allow for the non-invasive production of xeno-free conditioned media that may be used for autologous secretome therapy, thereby providing a GMP-compliant, scalable, quality-controlled protocol for the production of clinically usable therapeutic products.

[0010] The present invention also relates to conditioned medium containing one or more bioactive factors prepared using the methods of the present invention.

[0011] The present invention also relates to a conditioned medium containing one or more bioactive factors secreted by hair follicle-derived cells obtained from a subject.

[0012] The present invention also relates to compositions comprising the conditioned culture medium of the present invention and other diluents, excipients, or carriers.

[0013] Furthermore, the present invention relates to the use of the conditioned medium and compositions of the present invention for autologous or allogeneic secretome therapy.

[0014] Other and further aspects and features of the present disclosure will become apparent by reference to the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0015] For a better understanding of the various embodiments described herein, and to show more clearly how they may be carried into effect, reference is made, by way of example, to the accompanying drawings in which:

[0016] [Figure 1] A preferred example process flow diagram for preparing conditioned medium using hair follicles and an example use of such conditioned medium for treating hair loss is shown.

[0017] [Figure 2] Phase contrast images of hair follicles in growth culture medium on days 5 and 12 and hair follicle-derived cells grown from these follicles are shown.

[0018] [Figure 3] 1 shows the expression of keratinocyte markers in subpopulations of hair follicle-derived cells.

[0019] [Figure 4] 1 shows the expression of mesenchymal stem cell markers in a subpopulation of hair follicle-derived cells.

[0020] [Figure 5] 1 shows a cytokine array analysis using human antibodies, comparing the expression levels of analytes in hair follicle-derived conditioned medium according to one embodiment of the present invention with platelet-rich plasma prepared from the same subject.

[0021] [Figure 6] 1 shows an in vitro wound healing (scratch test) assay comparing human skin cells cultured in standard growth medium with human skin cells cultured in standard growth medium supplemented with hair follicle-derived conditioned medium, an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following description and the embodiments described herein are provided for the purpose of illustrating one or more examples of specific embodiments of various aspects of the present disclosure. These examples are provided for the purpose of illustrating the scope of the invention, and therefore are not intended to limit the scope of the invention. Well-known methods, procedures, and components have not been described in detail to avoid obscuring the exemplary embodiments described herein.

[0023] definition Unless otherwise stated, all words and phrases used herein include the meaning as used in the art unless a different meaning is clearly indicated or apparent from the context in which the word or phrase is used.

[0024] As used herein, the term "hair follicle" refers to the epidermal structure that originates at the surface of the epidermis and connects the hair shaft to the skin. Hair follicles contain cells and connective tissue and surround the hair root. The hair growth cycle is driven by a stem cell population present within the follicle. Hair follicles can be obtained using a variety of non-invasive methods, such as plucking from the scalp or follicular unit extraction.

[0025] As used herein, the term "anagen" refers to the active phase of the hair cycle characterized by rapid division and differentiation of cells, including stem cells, present in the bulge region of the outer root sheath.

[0026] As used herein, "stem cell" refers to a cell capable of self-renewal, i.e., a cell capable of sustaining multiple cell division cycles while remaining in a non-terminally differentiated state.

[0027] As used herein, the term "mesenchymal stem cells" refers to pluripotent cells that can differentiate into various cell lineages, including bone cells, adipocytes, and chondrocytes. Mesenchymal stem cells have been shown to possess a series of characteristic markers and also possess functions such as secreting immunomodulatory factors. According to certain preferred embodiments, the conditioned medium and compositions of the present invention comprise one or more bioactive factors secreted by mesenchymal stem cells derived from cultured hair follicles.

[0028] As used herein, the term "keratinocyte" refers to highly specialized cells present in the epidermal layer of skin. Keratinocytes play structural, protective, and immunoregulatory roles within the skin. Keratinocytes are responsible for repairing the epidermis, migrating to the wound after injury to secrete specific factors and initiate interactions between various cell types to facilitate a successful healing process. According to certain preferred embodiments, the conditioned medium and composition of the present invention contain one or more bioactive factors secreted by keratinocytes derived from cultured hair follicles.

[0029] As will be understood by those skilled in the art, the term "secretome" as used herein refers to any bioactive substance released by a cell. Secretomes may include growth factors, cytokines, and other proteins, nucleic acids, lipids, or carbohydrates. As part of the secretome, some bioactive molecules are secreted within cell membrane-bound vesicles, such as exosomes (see, for example, Reference [1]). Secretomes secreted from stem cells can exert a wide variety of important physiological functions by acting as molecular messengers responsible for communication between various types of cells. Various molecules contained in secretomes can generate signals that induce wound healing, cell proliferation, mobilization of extracellular matrix components, immunomodulation, and the like (see, for example, Reference [1]). Therefore, secretomes are believed to have a great potential to be effective in tissue repair and regeneration.

[0030] As used herein, the term "autologous" refers to cells, tissues, bodily fluids, etc. obtained from a subject and used in the same subject. For example, an autologous therapeutic product is one in which the subject from which the donor cells or donor tissue were obtained is the same subject as the recipient, and therefore the donor cells or donor tissue are genetically identical to the recipient.

[0031] As used herein, the term "allogeneic" refers to genetically distinct cells, tissues, bodily fluids, etc. For example, an allogeneic therapeutic product is one in which the cells or tissue are provided by a donor subject, and thus the cells or tissue are genetically distinct from the recipient.

[0032] As used herein, the term "substantially free" means that the presence of a particular component is not detectable by a known assay, or, if the presence of a particular component is detectable by a known assay, the content of that component is at least 99% or less.

[0033] As would be generally understood by one of ordinary skill in the relevant art, the term "conditioned medium" as used herein refers to a culture medium (or an extract, part, or fraction thereof) in which one or more cells of interest (e.g., hair follicle-derived cells) have been cultured. Culturing such cells results in the conditioning of the culture medium over time by secreting one or more molecules and / or bioactive factors into the extracellular space (e.g., into the culture medium). Conditioned medium may be unpurified or may be further processed. For example, the components of the conditioned medium may further contain one or more substances not secreted by the cells (e.g., additives, nutrients, etc., originally present in the growth culture medium). Alternatively, the conditioned medium is free of one or more substances not secreted by the cells, even in trace amounts.

[0034] As used herein, the term "cell culture" means growing cells under controlled conditions outside their natural environment.

[0035] As used herein, the terms "culture medium," "growth medium," and "growth culture medium" refer to a composition, often in liquid form, intended to support the growth and survival of cells or tissues outside their natural environment.

[0036] As used herein, the term "basal medium" refers to a synthetic medium without added additives, which may contain a buffer, one or more carbon sources, and one or more salts. Depending on the cell type and culture conditions, the basal medium may contain added additives and growth factors, including, but not limited to, additional buffers, amino acids, antibiotics, proteins, growth factors, and other nutrients essential for promoting the growth or survival of a particular cell of interest.

[0037] As used herein, the term "platelet-rich plasma" or "PRP" refers to a concentrate of a subject's own platelet-rich plasma, derived from whole blood and centrifuged to remove red blood cells. Platelet-rich plasma is being used by practitioners as an emerging treatment, often topically or in the form of an injectable solution, to accelerate the healing of various tissues throughout the body, including, but not limited to, tendon and ligament injuries, hair regeneration and skin rejuvenation, arthritis-related pain, retinal conditions, and other uses. In PRP treatments, methods for preparing PRP vary widely, depending on what is optimal for each specific clinical indication. For example, some procedures may further include an activation step, in which additives, such as, but not limited to, thrombin and calcium chloride, are added to the plasma and platelets to stimulate platelet granulation and release of growth factors into the medium. The resulting platelet-rich plasma is hereinafter referred to as "platelet lysate." The term "PRP derivative" is a generic term that encompasses platelet lysate or other forms of PRP that have been further processed after centrifugation or the addition of additives to improve cell culture efficacy.

[0038] As used herein, the term "effective amount" refers to an amount of a compound, molecule, component, composition, conditioned medium, or dilution thereof that is sufficient to achieve a desired effect (e.g., a therapeutic effect that inhibits injury or damage to one or more tissues in a subject).

[0039] As used herein, the term "lyophilization" refers to a method of removing water from a product by first freezing it and then placing it in an apparatus such as a vacuum pump, which converts the water from a solid phase directly to vapor, without passing through a liquid phase. This method is widely used to preserve perishable biological materials, either to extend their shelf life or to stabilize the material for transport purposes.

[0040] Preparation method Referring to FIG. 1, an exemplary process flow that may be used in various non-limiting embodiments of the present invention is shown, from sample collection to preparation of conditioned medium and use of such conditioned medium.

[0041] According to certain embodiments, the present invention provides a method for preparing a conditioned medium comprising one or more bioactive factors, the method comprising: obtaining hair follicles; conditioning the culture medium by culturing the hair follicles in the culture medium for a time sufficient for the hair follicle-derived cells to secrete one or more bioactive factors; and A step of recovering the obtained conditioned culture medium. The present invention provides a method comprising:

[0042] According to one embodiment of the present invention, hair follicles are obtained from a subject using a non-invasive method, such as plucking or follicular unit extraction. In a more specific embodiment, the harvested hair follicles may be cultured immediately. In another embodiment, the harvested hair follicles may be transported to a facility of interest in a transport composition described in WO2020 / 024045A1. In yet another embodiment, the harvested hair follicles may be cryopreserved in a cryopreservation composition described in WO2020 / 024045A1 for various periods of time, followed by thawing and culture.

[0043] PRP-derived additives have been used in in vitro cell and tissue culture as an alternative to animal serum and serum-free additives, as well as directly as a safe and effective treatment for numerous clinical applications (see, e.g., Reference [2]). Therefore, in a more specific embodiment, the culture medium used to prepare the conditioned medium contains PRP or a PRP derivative (e.g., platelet lysate). In a preferred embodiment, the culture medium used to prepare the conditioned medium contains about 1% to about 30% w / w platelet lysate, preferably about 5% to about 25% w / w platelet lysate, and more preferably about 20% w / w platelet lysate.

[0044] Developing an autologous conditioned medium therapy can enhance clinical safety and meet regulatory and commercial requirements for clinical products. Thus, in certain embodiments, the PRP or PRP derivative (e.g., platelet lysate) is autologous PRP or an autologous PRP derivative obtained from the subject from whom the hair follicles used to condition the culture medium were obtained. According to certain embodiments, PRP is prepared by drawing whole blood from the subject and centrifuging it for an appropriate period of time under conditions standard in the art. In one embodiment, PRP may be activated by adding chemicals, such as, but not limited to, thrombin and calcium chloride, which can enhance the release of growth factors into the composition and form platelet lysate. In a preferred embodiment, the concentration of the chemicals (e.g., thrombin and / or calcium chloride) that activate PRP is about 20 mM to about 30 mM. Alternatively, according to certain embodiments, the culture medium may contain additives such as blood substitutes, human recombinant growth media, or other commercially available xeno-free nutrients.

[0045] According to certain embodiments, no animal serum or other animal components are used in the preparation of the conditioned medium. In one embodiment, large numbers of human hair follicles are cultured in a culture medium that is largely autologous and completely xeno-free.

[0046] According to certain embodiments, the culture medium for culturing hair follicles may include, but is not limited to, a basal medium known in the art to which the present invention pertains. The basal medium may be prepared by artificial synthesis, or a commercially available medium may be used. Examples of commercially available media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle Medium (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), and modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium). In some embodiments, the culture medium includes KnockOut Dulbecco's Modified Eagle Medium / F-12, MEM, BME, platelet lysate, or other xeno-free media known in the art.

[0047] According to certain embodiments, the culture medium may contain additional growth factors in amounts effective to stimulate cell growth and proliferation, including, but not limited to, basic fibroblast growth factor (b-FGF). In further embodiments, the culture medium may further contain additional antibiotics, such as penicillin-streptomycin, in amounts standard in the art to prevent bacterial contamination.

[0048] In one embodiment, the culture medium comprises components selected from KnockOut Dulbecco's Modified Eagle's Medium / F-12, MEM, BME, or other xeno-free medium known in the art, autologous platelet lysate at a concentration of about 5% to about 25% w / w, penicillin and / or streptomycin, and any combination thereof.

[0049] According to certain embodiments, the hair follicles are cultured under conditions optimal for stimulating cell proliferation. In some embodiments, the hair follicles are cultured at a temperature of about 20°C to about 50°C. In some preferred embodiments, the hair follicles are cultured at a temperature of about 30°C to about 40°C, with about 37°C being more preferred.

[0050] According to certain embodiments, the hair follicles are maintained at about 2% to about 10% CO2, preferably at about 5% CO2.

[0051] According to certain embodiments, hair follicles may be cultured in growth culture medium for about 1 week to about 6 weeks, preferably about 4 weeks. Referring now to Figure 2, phase contrast microscopy images are shown demonstrating proliferation of hair follicle-derived cells from the outer root sheath of hair follicles in culture from days 5 to 12.

[0052] According to certain embodiments, culturing hair follicles in a growth culture medium according to standard procedures known to those skilled in the art results in a heterogeneous cell population, which may include, but is not limited to, keratinocytes, mesenchymal stem cells, fibroblasts, hair-associated multipotent cells, dermal papilla cells, and other primary somatic or primary stem cells. For example, referring to FIG. 3, immunofluorescence staining of expanded hair follicle-derived keratinocytes demonstrates significant expression of basal layer keratinocyte markers KRT14 and KRT5. Another example of a cell type is shown in FIG. 4, which shows immunofluorescence staining of mesenchymal stem cells isolated from hair follicles for standard mesenchymal-associated markers known in the art. These mesenchymal stem cells express the typical immunophenotypic profile of mesenchymal stem cells (CD90+CD105+CD73+CD44+ and CD45-CD31-). The culture of various cell types and the expression of paracrine factors can affect the therapeutic efficacy of the conditioned medium described herein or compositions comprising the conditioned medium.

[0053] According to certain embodiments, the culture medium is conditioned by culturing hair follicles in the culture medium until the follicles reach about 80% to about 90% confluence. In one embodiment, the conditioned medium is collected at regular intervals from about 12 hours to about 96 hours from the start of the culture.

[0054] According to certain embodiments, the conditioned medium is further processed. In one embodiment, the conditioned medium is purified by centrifuging the conditioned medium followed by filtration to remove cellular debris. In a more specific embodiment, the conditioned medium is filtered using a 0.22 μm filter to ensure removal of cellular material. In another embodiment, the conditioned medium is cryopreserved, frozen, or lyophilized. In a more specific embodiment, an effective amount of a lyophilizing agent, such as sucrose, is added to the conditioned medium to act as a protein stabilizer and protect the conditioned medium from the lyophilization process. Determining the optimal lyophilizing agent and its effective amount is within the ordinary knowledge and skill of one of ordinary skill in the art. In a further embodiment, the lyophilized conditioned medium is suspended in a suspension mixture comprising one or more of saline, hyaluronic acid, silicone gel, petrolatum, PRP, a PRP derivative, platelet lysate, and a pharmaceutically acceptable excipient.

[0055] According to one preferred embodiment, the method for preparing a conditioned medium containing one or more bioactive factors comprises the steps of: non-invasively harvesting hair follicles from a subject by scalp plucking or follicular unit extraction; culturing the hair follicles in xeno-free culture medium or autologous PRP, or a combination thereof, to induce cell proliferation; and Once the cultured cells reach confluence, the conditioned medium is collected. Including, Further processing the conditioned medium by centrifugation, filtration, lyophilization, or other processes to remove cellular material and ensure the purity and stability of the final product. may also include:

[0056] Conditioned culture medium According to certain embodiments, the present invention provides a conditioned medium comprising one or more bioactive factors secreted by hair follicle-derived cells obtained from a subject. In some embodiments, such a conditioned medium is prepared using the preparation methods described herein.

[0057] According to certain embodiments, the one or more factors secreted by the hair follicle-derived cells may include regenerative factors, which may include proteins (e.g., growth factors, cytokines, chemokines), nucleic acids (e.g., miRNA), polysaccharides (e.g., hyaluronic acid), and / or combinations thereof, which are either bound to the interior of extracellular vesicles (e.g., exosomes) or are not contained in extracellular vesicles.

[0058] According to certain embodiments, the one or more bioactive factors include, but are not limited to, hyaluronic acid, elastin, HAPLN1, collagen (e.g., COL1A1, COL2A1, COL3A1), keratin (e.g., KRT5, KRT19), fibronectin, EMILIN1, KGF, PDGF, bFGF, TGF-β1, HGF, EGF, VEGF, CCL19, sAXL, SDF-1, VCAM-1, MCP-1, IGF-1, GDNF, BDNF, NRG2, PDGF, interleukins (e.g., IL-1, IL-2, IL-6), or any combination thereof, and the concentrations of these bioactive factors vary depending on the subject and range from 0 μg / mL to 1000 μg / mL. Referring now to FIG. 5, an antibody-based cytokine array analysis is shown, illustrating the expression levels of various bioactive factors detected in conditioned medium according to one non-limiting embodiment.

[0059] According to certain embodiments, the present invention further provides a composition comprising the conditioned medium disclosed herein and one or more of saline, hyaluronic acid, PRP, a PRP derivative, platelet lysate, and pharmaceutically acceptable excipients, carriers, and diluents. In a further embodiment, the composition of the present invention is formulated into a dosage form selected from an injection, an oil suspension, a hydrogel, a nanogel, an ointment, a cream, a serum preparation, an emulsion, a spray, a patch, a gel, a drop, or any combination thereof. In a more specific embodiment, the present invention provides a pharmaceutical or cosmetic composition comprising the conditioned medium, extracellular vesicles, autologous PRP, hyaluronic acid, or a combination thereof.

[0060] According to certain embodiments, the conditioned medium or composition of the present invention is substantially free of animal serum or animal components. Animal serum and other animal-derived components are widely used in cell and / or tissue culture as a source of growth factors, hormones, amino acids, lipids, and other nutrients. However, animal serum and other animal-derived components raise biosafety concerns and contribute to batch-to-batch variability. While most prior art secretome therapies involve switching to serum-free culture medium before secretome harvest, concerns remain due to the use of animal products at some stage in the cell culture process. The introduction of contaminants during manufacturing and the potential for contamination of the final therapeutic product pose a major safety concern when transitioning to clinical applications.

[0061] According to certain embodiments, the conditioned medium or composition of the present invention contains additional soluble components, including, but not limited to, growth factors in the growth culture medium, intended to stimulate the growth and proliferation of hair follicle-derived cells cultured therein. In one embodiment, such growth factors are derived from PRP or PRP derivatives contained in the culture medium. In another embodiment, growth factors are added to the culture medium, including, but not limited to, recombinant human basic fibroblast growth factor, as well as other recombinant, synthetic, and human-derived growth factors, at concentrations of about 0.01 ng / ml to about 10 ng / ml.

[0062] According to certain embodiments, the conditioned medium or composition of the present invention is substantially free of whole cells or cell debris. In one embodiment, the conditioned medium is a cell-free secretome product prepared from a heterogeneous primary cell population cultured from human hair follicles.

[0063] According to certain embodiments, the conditioned medium or composition of the present invention is a home-made product derived from the subject.

[0064] According to one preferred embodiment, the conditioned medium or composition of the present invention is a xeno-free, homemade product prepared by culturing human hair follicles in xeno-free conditions to stimulate the growth of hair follicle-derived cells. In another preferred embodiment, the conditioned medium or composition of the present invention is prepared by culturing human hair follicles in PRP or a PRP derivative to stimulate the growth of hair follicle-derived cells.

[0065] According to certain embodiments, the conditioned medium or compositions of the present invention may be used for therapeutic or cosmetic purposes, including wound healing at a target tissue site, stimulating soft or connective tissue repair / regeneration at a target tissue site (e.g., hair regrowth, skin rejuvenation, slowing or arresting signs of skin aging, or treating joint-related conditions), or delivering one or more bioactive factors to a target tissue site. Referring now to Figure 6, a scratch assay comparing wound healing of human skin cells cultured in standard growth medium with wound healing of human skin cells cultured in standard growth medium supplemented with a conditioned medium according to a preferred embodiment is shown.

[0066] In one embodiment, the conditioned medium or composition of the present invention is administered in a therapeutically effective amount to a subject or cells or tissues derived from a subject for a suitable period of time. In a preferred embodiment, the conditioned medium or composition of the present invention is used for autologous treatment in a subject from which hair follicles have been harvested. In another embodiment, the conditioned medium or composition of the present invention may be used for allogeneic treatment in a subject, in which the conditioned medium is prepared from hair follicles of a healthy donor.

[0067] According to certain embodiments, the conditioned medium may be used directly and fresh after preparation or after lyophilization. In one embodiment, the lyophilized conditioned medium may be stored at room temperature for about 12 weeks or at about 4° C. for up to about 24 months. In another embodiment, the lyophilized conditioned medium may be mixed with a solid carrier, a liquid carrier such as PRP, hyaluronic acid, saline, or the like, a cream for topical application, or a serum for injectable use.

[0068] kit According to certain embodiments, the present invention provides a kit comprising a culture medium suitable for culturing hair follicles and instructions to instruct a subject how to prepare the hair follicle-derived conditioned medium. [Example]

[0069] The present invention will be further described by, but not limited to, the following examples, which are provided to facilitate understanding of the present disclosure, but which do not limit the scope of the present invention in any way and should not be construed as limiting the scope of the present invention.

[0070] The following examples do not include detailed descriptions of well-established conventional methods known to those skilled in the art.

[0071] Example 1 - Preparation of conditioned medium by culturing hair follicles in autologous platelet lysate This example describes a preferred method for preparing a conditioned medium containing one or more bioactive factors using hair follicles, in accordance with one embodiment of the present invention.

[0072] Hair follicles are rinsed thoroughly with antibiotic-antimycotic solution, cut 1-2 mm distal to the outer root sheath, and seeded onto culture vessels pre-coated with CELLstart substrate. The follicles are then incubated at 37°C in 5% CO2 and 5% O2 for 2-4 weeks.

[0073] A culture growth medium consisting of 20% autologous platelet lysate diluted in basal medium is added to the seeded hair follicles, and the culture growth medium is changed every two days to maintain cell viability.

[0074] Once the hair follicle-derived cells reach 70-90% confluence, collect the conditioned culture medium, centrifuge it at 300 x g for 5 min, and filter-sterilize the supernatant through a disposable 0.22 µm sterile filter system.

[0075] The resulting solution is then aliquoted into sterile vials and stored at 4°C for immediate use or lyophilized for easy long-term storage.

[0076] Example 2 - Ex vivo wound healing assay comparing hair follicle-derived conditioned medium with standard growth medium This example describes an ex vivo wound healing assay to demonstrate the effect of hair follicle-derived conditioned medium on wound healing compared to the use of standard growth medium alone as a control.

[0077] The target skin cells were cultured in 12-well plates until confluent. Once confluent, wounds were created by scraping the cells off the surface of the plate with a scalpel. This procedure resulted in artificial wounds with no cells present at the scraped area.

[0078] The artificially wounded skin cells were then cultured in standard growth medium, known to promote proliferation and migration, or in this standard growth medium supplemented with 10% conditioned medium prepared according to the method described in Example 1.

[0079] Artificially wounded skin cells were observed to measure wound closure, cell migration, and cell proliferation. As shown in Figure 6, skin cells cultured in 10% conditioned medium after artificial wound creation showed approximately 3.5-fold increased wound closure after only approximately 48 hours of culture, due to enhanced cell proliferation and migration, compared to control skin cells cultured in standard growth medium alone. The results of this wound healing assay demonstrated that hair follicle-derived conditioned medium improved wound healing.

[0080] Example 3 - Hair follicle-derived conditioned medium as a potential treatment for male pattern baldness This example describes a clinical trial to evaluate the efficacy of hair follicle-derived conditioned medium for the treatment of hair loss conditions, such as male pattern baldness.

[0081] The clinical trial will select subjects diagnosed with male pattern baldness using the following inclusion criteria: Male subjects with a clinical diagnosis of male pattern baldness. Subjects must consent to the following sample collection: Plucking of at least 20 hair follicles from the scalp at the back of the head A total of 30 ml of blood was collected

[0082] By collecting hair follicles and a blood sample from a subject, an autologous hair follicle-derived conditioned culture medium can be prepared according to the preparation method described in Example 1.

[0083] The resulting conditioned medium is lyophilized. The lyophilized conditioned medium is suspended in PRP, hyaluronic acid, saline, or a suitable solvent or carrier.

[0084] Subjects will receive an effective dose of conditioned medium topically or intradermally at the treatment site once a month for five months.

[0085] Throughout the clinical trial, baseline examinations will include trichogram analysis to assess hair density and anagen induction, and clinical photography and ultrasonography to assess skin thickness and echogenicity.

[0086] Example 4 - Hair follicle-derived conditioned medium as a therapeutic candidate for improving wound healing and inhibiting scar tissue This example describes a clinical trial to evaluate the effectiveness of hair follicle-derived conditioned medium in improving and accelerating wound healing after elective plastic surgery.

[0087] People planning to have an abdominoplasty (tummy tuck) to remove excess skin and fat from the abdomen. Female subjects who have a scheduled abdominoplasty surgery date. Subjects must consent to the following sample collection: Plucking of at least 20 hair follicles from the occipital scalp one month before surgery

[0088] By collecting hair follicles from a subject, an autologous hair follicle-derived conditioned culture medium can be prepared according to the preparation method described in Example 1.

[0089] The resulting conditioned medium is freeze-dried, and the freeze-dried conditioned medium is suspended in hyaluronic acid.

[0090] An effective dose of conditioned medium is administered topically to one half of the abdominoplasty site of each subject once daily for seven days. Hyaluronic acid is administered topically to the other half of the abdominoplasty site once daily for seven days. In this way, a half-side test can be performed on each subject's treatment and control sites.

[0091] Clinical photography and ultrasound to assess skin thickness, surface topography, scar tissue formation and healing time will be performed as baseline examinations throughout the clinical trial.

[0092] Those skilled in the art will appreciate that additional alternative implementations and variations are possible, and that the above examples are merely illustrative of one or more implementations. Accordingly, the scope of the present invention is limited only by the claims appended hereto.

[0093] interpretation For purposes of this application, the terms "at least one of X, Y, and Z" or "one or more of X, Y, and Z" may be interpreted to refer to X only, Y only, Z only, or any combination of two or more of X, Y, and Z (e.g., XYZ, XYY, YZ, ZZ).

[0094] In this application, each component may be described as being "configured to" or "capable of" performing one or more functions. Generally, a component configured to perform a function or capable of performing a function is interpreted as being configured to perform or capable of performing that function, suitable to perform that function, adapted to perform that function, operative to perform that function, or otherwise capable of performing that function.

[0095] As used herein, terms such as "one embodiment," "embodiment," "implementation," "variant," and the like refer to an embodiment, implementation, or variant described herein that may include a particular aspect, feature, structure, or characteristic, but not all embodiments, implementations, or variants necessarily include that aspect, feature, structure, or characteristic. Furthermore, these phrases may, but do not necessarily, refer to the same embodiment(s) mentioned elsewhere in this specification. Furthermore, when a particular aspect, feature, structure, or characteristic is described with respect to a particular embodiment, it is within the knowledge of one skilled in the art that such module, aspect, feature, structure, or characteristic also affects or is related to other embodiments, whether or not explicitly stated. In other words, any module, component, or feature may be combined with other components or features of other embodiments unless there is a clear inconsistency, a substantial incompatibility, or unless specifically excluded.

[0096] It should also be noted that the claims may be written to exclude optional elements. This consideration presupposes the use of exclusive terms such as "solely" and "only" in connection with the recitation of claim elements and the use of "negative" limitations. The terms "preferably," "preferred," "preferred," "optionally," and "may," and similar terms, are used to indicate that a recited item, state, or step is an optional (but not required) feature of the invention.

[0097] Additionally, the terms "comprises," "comprising," "includes," and / or "including" specify the presence of features, steps, operations, components, and / or ingredients described herein, but do not exclude the presence or addition of one or more other features, steps, operations, components, ingredients, and / or groups thereof.

[0098] The singular forms "a," "an," and "the" include plural references unless expressly stated otherwise. The term "and / or" means any one, any combination, or all of the items with which this term is associated. The term "one or more" can be readily understood by those of ordinary skill in the art, especially by reference to the context in which the term is used.

[0099] For purposes of providing a detailed description, and for other purposes, those skilled in the art will understand that all ranges described herein encompass all possible subranges and combinations thereof, as well as the individual numerical values ​​(especially integers) comprising the range. The ranges described herein include each and every specific numerical value, integer, fractional value, or unit within the range. Any range described herein can be divided into at least three equal parts, three equal parts, four equal parts, five equal parts, ten equal parts, etc., and it will be readily understood that the invention can be practiced within such divided ranges. For example, any range described herein can be easily divided into three equal parts, such as a low range, a midrange, and a high range, but is not limited thereto. References 1. Wangler, S., Kamali, A., Wapp, C. et al. Uncovering the secretome of mesenchymal stromal cells exposed to healthy, traumatic, and degenerative intervertebral discs: a proteomic analysis. Stem Cell Res Ther 12, 11 (2021). 2. Burnouf, T., Strunk, D., Koh, M. B., & Schallmoser, K. (2016). Human platelet lysate: replacing fetal bovine serum as a gold standard for human cell propagation?. Biomaterials, 76, 371-387. 3. Walter MN, Wright KT, Fuller HR, MacNeil S, Johnson WE. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res. 2010 Apr 15;316(7):1271-81. 4. Topouzi, Helena, et al. Harnessing the secretome of hair follicle fibroblasts to accelerate ex vivo healing of human skin wounds. Journal of Investigative Dermatology 140.5 (2020): 1075-1084.

Claims

1. 1. A method for preparing a conditioned medium containing one or more physiologically active factors, comprising: obtaining non-invasively harvested hair follicles from a subject; conditioning the culture medium by culturing the hair follicles in the culture medium for a time sufficient for the hair follicle-derived cells to secrete one or more bioactive factors; and A step of recovering the obtained conditioned culture medium. A method comprising:

2. 10. The method of claim 1, wherein the culture medium comprises platelet-rich plasma (PRP), a PRP derivative, a platelet lysate, or about 1% to about 30% w / w platelet lysate.

3. 3. The method of claim 2, wherein the PRP, PRP derivative, or platelet lysate is autologous PRP, autologous PRP derivative, or autologous platelet lysate obtained from the subject from whom the hair follicles were obtained.

4. 4. The method of claim 2 or 3, wherein the platelet lysate is prepared by activating PRP with thrombin, calcium chloride, or any combination thereof, at a concentration of about 20 mM to about 30 mM.

5. 5. The method of any one of claims 1 to 4, wherein the culture medium comprises KnockOut Dulbecco's Modified Eagle's Medium / F-12, Minimum Essential Medium, Eagle's Basal Medium, Xeno-Free Medium, penicillin, streptomycin, about 0.01 ng / mL to about 10 ng / mL of a growth factor or basic fibroblast growth factor, or any combination thereof.

6. The method according to any one of claims 1 to 5, wherein the hair follicle-derived cells are keratinocytes, mesenchymal stem cells, fibroblasts, hair-associated multipotent cells, dermal papilla cells, hair follicle-derived stem cells, hair follicle-derived somatic cells, or any combination thereof.

7. The method of any one of claims 1 to 6, wherein the hair follicles are cultured to about 80% to about 90% confluence.

8. removing cellular debris by centrifugation and filtration of the conditioned medium; Cryopreserving, freezing or lyophilizing the conditioned culture medium; suspending the lyophilized conditioned medium in a suspension mixture comprising one or more of saline, hyaluronic acid, silicone gel, petrolatum, PRP, a PRP derivative, platelet lysate, and a pharmaceutically acceptable excipient. The method of any one of claims 1 to 7, further comprising at least one of:

9. A conditioned culture medium prepared using the method according to any one of claims 1 to 8.

10. A conditioned medium containing one or more physiologically active factors secreted by hair follicle-derived cells obtained non-invasively from a subject.

11. 11. The conditioned medium of claim 9 or 10, wherein the one or more bioactive factors comprise a growth factor, a cytokine, a chemokine, a small peptide, a nucleic acid, an extracellular matrix molecule, an extracellular vesicle, a factor capable of mediating a physiological process, or any combination thereof.

12. 12. The conditioned medium of claim 11, wherein the physiological process comprises wound healing, cell proliferation, angiogenesis, an anti-inflammatory response, or any combination thereof.

13. 13. The conditioned culture medium of any one of claims 9 to 12, wherein the one or more bioactive factors comprise hyaluronic acid, elastin, HAPLN1, collagen (e.g., COL1A1, COL2A1, COL3A1), keratin (e.g., KRT5, KRT19), fibronectin, EMILIN1, KGF, PDGF, bFGF, TGF-β1, HGF, EGF, VEGF, CCL19, sAXL, SDF-1, VCAM-1, MCP-1, IGF-1, GDNF, BDNF, NRG2, PDGF, interleukin (e.g., IL-1, IL-2, IL-6), or any combination thereof, at a concentration of about 0 μg / mL to about 1000 μg / mL.

14. A conditioned culture medium according to any one of claims 9 to 13, which is substantially free of whole cells, cell debris and / or xenogeneic components.

15. A composition comprising the conditioned culture medium of any one of claims 9 to 14 and one or more of saline, hyaluronic acid, PRP, a PRP derivative, a platelet lysate, and a pharmaceutically acceptable excipient, carrier, and diluent.

16. The composition of claim 15, wherein the PRP, PRP derivative, or platelet lysate is autologous PRP, autologous PRP derivative, or autologous platelet lysate obtained from the subject from whom the hair follicle-derived cells used to condition the conditioned medium were obtained.

17. 17. The composition according to claim 15 or 16, which is formulated into a dosage form selected from an injection, an oily suspension, a hydrogel, a nanogel, an ointment, a cream, a serum preparation, an emulsion, a spray, a patch, a gel, a drop, and any combination thereof.

18. Use of the conditioned medium according to any one of claims 9 to 14 or the composition according to any one of claims 15 to 17 for therapeutic or cosmetic treatment.

19. 19. The use of claim 18, wherein the therapeutic or cosmetic treatment comprises stimulating soft or connective tissue repair at a target tissue site, hair regrowth, skin rejuvenation, slowing or arresting the signs of skin aging, or treating a joint-related condition.

20. 20. The use according to claim 18 or 19, wherein the target tissue site is the tissue site of the subject from which the hair follicle-derived cells used to condition the conditioned medium were obtained.

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