Conditioned culture medium from human hair follicle and preparation method of conditioned culture medium

Abstract

The present invention provides a method of preparing a hair follicle-derived conditioned medium comprising one or more bioactive factors wherein the hair follicles are obtained in a non-invasive manner from a subject, the one or more bioactive factors are cultured in a culture medium sufficient to cause the hair follicle-derived cells to secrete the one or more bioactive factors to condition the culture medium for a duration of time. The present invention provides a hair follicle-derived conditioned medium comprising one or more bioactive factors secreted by hair follicle-derived cells non-invasively obtained from a subject. The invention provides a composition comprising the conditioned medium. The invention provides the use of the conditioned media and compositions for therapeutic or cosmetic treatment.

Description

Technical Field

[0001] This specification generally relates to secretome-based therapies, and more particularly to conditioned media prepared non-invasively using hair follicles. Background Art

[0002] Secretome-based therapies have received much attention in the therapeutic and cosmetic fields for treating various conditions, including hair loss, skin conditions, and sports-related injuries. These effects are mediated by the action of a range of secreted bioactive factors, such as cytokines and growth factors, which are collectively referred to as the "secretome". Although the need for highly invasive tissue collection methods and poor product scalability are major limitations that have been overlooked in the prior art, a large body of evidence from previous studies confirms the broad therapeutic promise of harnessing the secretome of stem cells.

[0003] In the prior art, different tissues where stem cells are located have been studied for secretome therapy. These include bone marrow, adipose tissue, amniotic fluid, or umbilical cord, all of which are collected by highly invasive methods. Examples of prior art conditioned media therapeutics available on the market include BabyStem from bone marrow TM , BioCM from amniotic fluid TM and several compositions from adipose tissue, such as AAPE TM , Exomide TM , TheraStem TM , Stem C’rum TM , etc. In addition to tissue invasiveness, all of the said products are allogeneic therapies harvested from continuously expanded and cultured cells, with little or no public information about the cellular and donor sources of these cells. It has been well established that cells can lose or acquire various intrinsic properties over long-term passaging, including alterations in the proteome profile. We now know that the content of the secretome varies widely between donors, which poses the greatest challenge to allogeneic therapies. Although secretome therapies generally mitigate the safety risks inherent in administering large numbers of live stem cells, allogeneic sources remain an obstacle to the safety of these therapies.

[0004] Another factor that is overlooked in prior art secretome therapies is the inability to exclude the presence of animal (xenogeneic) serum residues in the composition of the final product. Animal-derived components, such as fetal bovine serum, are commonly used to culture cells and tissues in culture. This raises reliability and biosafety issues. Although most prior art secretome therapies switch to xeno-free medium shortly before conditioned medium collection, the use of animal products at any stage during the cell culture process remains problematic. The main concern of using animal components is the introduction of contaminants into the process, and therefore potentially into the final therapeutic product, which makes these animal components unsuitable for clinical or therapeutic use. Therefore, the use of xeno-free reagents throughout the secretome generation process is necessary to ensure clinical safety of human subjects.

[0005] The use of completely serum-free media has many advantages, including consistency of preparation and biosafety. However, it can result in suboptimal growth and cell metabolism, and there is an unmet need for alternative methods to enrich for secretome composition in a manner that is also safe for human use.

[0006] Therefore, new secretome therapies and preparation methods are needed to overcome the limitations of existing secretome therapies. The present invention discloses conditioned medium that can be used for autologous secretome therapies, which is prepared non-invasively using hair follicles and expands cells using a xeno-free system. Summary of the invention

[0007] The present invention relates to a method for non-invasively producing a conditioned medium containing one or more bioactive factors using hair follicles. Although the role of stem cells located in the dermal papilla of hair follicles in immunomodulation and wound healing has been widely studied, these cells have historically been obtained using invasive means, including through follicular unit extraction (FUE, a technique for hair transplantation). In various aspects, the present invention is based on the following discovery: cells located in the upper region of the hair follicle (which can be continuously obtained non-invasively) can be used and are a high-quality source for producing secretome therapies. In addition, not only can cells located in the upper region of the hair follicle be continuously obtained non-invasively, but they are also easier to culture directly from the hair follicle and do not require digestive enzymes. Compared with other stem cell tissue resources, human hair follicles in the growth phase state are rich tissue resources that can be easily and non-invasively obtained throughout adulthood. There are various heterogeneous cell types in the outer root sheath of the hair follicle, including endogenous stem cell populations responsible for hair growth and development. Therefore, it is of great value to use this tissue resource for the production of secretome therapies.

[0008] According to one embodiment, the preparation method of the present invention includes obtaining hair follicles by non-invasive means, culturing the hair follicles in a growth medium (such as but not limited to autologous platelet lysate) for a suitable period of time such that cells derived from the hair follicles secrete one or more molecules into the growth medium, thereby conditioning the medium, and collecting the conditioned medium for further processing or immediate use.

[0009] In certain embodiments, the preparation method of the present invention non-invasively produces a xeno-free conditioned medium that can be used in autologous secretome therapy, thereby providing a GMP-ready, scalable, and quality-controlled protocol for generating clinically ready-to-use therapeutic products.

[0010] The present invention also relates to a conditioned medium prepared by the method of the present invention, which contains one or more bioactive factors.

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

[0012] The present invention further relates to a composition that contains the conditioned medium of the present invention and other diluents, excipients, or carriers.

[0013] The present invention also relates to the use of the conditioned medium and composition of the present invention in autologous or allogeneic secretome therapy.

[0014] Other and further aspects and features of the present disclosure will become apparent during the following discussion and by reference to the accompanying drawings. Description of the Drawings

[0015] To better understand the various embodiments described herein and to more clearly show how they may be implemented, reference is now made, by way of example only, to the accompanying drawings, in which:

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

[0017] Figure 2 Phase contrast images showing the outgrowth of hair follicles and their derived cells from the hair follicles in a growth medium at day 5 and day 12 are shown.

[0018] Figure 3 Expression of keratinocyte markers in a subset of cells derived from hair follicles is shown.

[0019] Figure 4 Expression of mesenchymal stem cell markers in a subset of cells derived from hair follicles is shown.

[0020] Figure 5Shows a human antibody cytokine array analysis that shows, as one embodiment of the present invention, the levels of analytes in the hair follicle-derived conditioned medium compared to platelet-rich plasma prepared from the same subject.

[0021] Figure 6 Shows one embodiment of the present invention, an in vitro wound healing (scratch test) assay of human skin cells cultured in standard growth medium compared to standard growth medium with hair follicle-derived conditioned medium. Detailed Description

[0022] The following description and the embodiments described therein are provided by way of one or more examples of specific embodiments that illustrate various aspects of the present disclosure. These examples are provided for purposes of explanation and 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 aspects described herein.

[0023] Definition

[0024] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless there is a contrary indication either explicit or obvious from the context in which the phrase is used.

[0025] As used herein, the term "hair follicle" refers to an epidermal structure that originates at the epidermal surface and anchors the hair shaft to the skin. It contains cells, connective tissue, and surrounds the hair root. The cyclic growth of hair is driven by a population of stem cells present within the hair follicle. Hair follicles can be obtained using a variety of non-invasive methods, such as plucking, follicular unit extraction, or other methods.

[0026] As used herein, the term "anagen" refers to the active phase of the hair growth cycle, which is characterized by rapid cell division and differentiation of cells, including stem cells present in the bulge region of the outer root sheath.

[0027] As used herein, "stem cell" refers to a cell that has the ability to self-renew (i.e., the ability to maintain multiple cell division cycles while remaining in a non-terminally differentiated state).

[0028] As used herein, the term "mesenchymal stem cell" is a pluripotent cell that can differentiate into multiple cell lineages, including osteocytes, adipocytes, and chondrocytes. It is found to have a characteristic set of markers and has additional functions, including the secretion of immunomodulatory factors. According to certain preferred embodiments, the conditioned medium and compositions of the present invention contain one or more bioactive factors secreted by mesenchymal stem cells from cultured hair follicles.

[0029] As used herein, the term "keratinocyte" refers to highly specialized cells within the epidermal layer of the skin. It has a structural, protective, and immunomodulatory role within the skin. It is responsible for restoring the epidermis after injury by migrating to the wound and secreting specific factors to initiate interactions between different cell types for a successful healing process. According to certain preferred embodiments, the conditioned media and compositions of the present invention contain one or more bioactive factors secreted by keratinocytes from cultured hair follicles.

[0030] As used herein, as would be generally understood by one of ordinary skill in the art, the term "secretome" refers to the sum total of all bioactive substances released by a cell. These can include growth factors, cytokines, and other proteins, nucleic acids, lipids, or hydrocarbons. As part of the secretome, some bioactive molecules are secreted within membrane-bound vesicles, such as exosomes (see, for example, reference [1]). The secretome from stem cells can play a wide range of important physiological functions by acting as molecular messengers to transmit signals between different cell types. The various molecules within the secretome can signal for wound healing, cell proliferation, recruitment of extracellular matrix components, immunomodulation, etc. (see, for example, reference [1]). Thus, the secretome is thought to have great potential for tissue repair and regeneration.

[0031] As used herein, the term "autologous" refers to cells, tissues, body fluids, etc. obtained from the same subject; for example, an autologous therapeutic product is one in which the donor cells or tissues are taken from the same subject who is the recipient of the product, such that the donor cells or tissues are genetically identical to the recipient.

[0032] As used herein, the term "allogeneic" refers to cells, tissues, body fluids, etc. that are genetically dissimilar; for example, an allogeneic therapeutic product is one in which the cells or tissues are donated by a donor subject and are thus genetically different from the recipient of the product.

[0033] As used herein, the term "substantially free of" means that the presence of a particular component is not detected using known assays, or if detected, it is present only in an amount that is at least 99% free of that component.

[0034] As used herein, one of ordinary skill in the art would typically understand the term "conditioned medium" to be a medium (or an extract, portion, or fraction thereof) in which one or more target cells (e.g., cells derived from hair follicles) have been cultured. Culturing of the cells can result in the secretion of one or more molecules and / or bioactive factors into the extracellular space (e.g., into the medium) over a period of time, thereby conditioning the medium. The conditioned medium can be kept unpurified or further processed. For example, the components of the conditioned medium can further include one or more substances that are not secreted from the cells (e.g., additives and nutrients included in the initial growth medium). Alternatively, the conditioned medium does not include one or more substances that are not secreted from the cells or includes them in trace amounts.

[0035] As used herein, the term "cell culture" refers to cells grown under controlled conditions outside of their natural environment.

[0036] As used herein, the terms "medium", "growth medium", and "growth culture medium" refer to compositions designed to support the growth and survival of cells or tissues outside of their natural environment and are typically in liquid form.

[0037] As used herein, the term "basal medium" refers to a synthetic medium that may contain buffers, one or more carbon sources, and salts without added supplements. Depending on the cell type and culture conditions, the basal medium can be supplemented with additives and growth factors, including but not limited to additional buffers, amino acids, antibiotics, proteins, growth factors, and other nutrients necessary to promote the growth or survival of specific target cells.

[0038] As used herein, the term "platelet-rich plasma" or "PRP" refers to a concentration of a subject's own plasma that is rich in platelets and is derived from whole blood that has been centrifuged to remove red blood cells. Those skilled in the art have used it as an emerging therapy, typically in the form of topical application or injection, to accelerate the healing of different tissues in the body, such as but not limited to tendon and ligament injuries, hair and skin rejuvenation, arthritis-related pain, retinal conditions, and other applications. PRP therapies vary greatly in their preparation methods, depending on the optimal scenario for each specific clinical indication. For example, some procedures can further include an activation step, by which additives (such as but not limited to thrombin or calcium chloride) are added to the plasma and platelets to stimulate granulation of the platelets and release growth factors into the medium, which is thereafter referred to as "platelet lysate". The term "PRP derivative" refers to a general term covering platelet lysate or any other form of PRP that has been further processed or had additives added after centrifugation to enhance cell culture efficacy.

[0039] As used herein, the term "effective amount" refers to the 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, controlling damage or injury to one or more tissues experienced by a subject).

[0040] As used herein, the term "lyophilization" refers to the process of removing water from a product after the product has been frozen and placed in a near-vacuum device, thereby allowing the water to change directly from the solid phase to the vapor phase without passing through the liquid phase. It is a method commonly used to preserve perishable biological materials, extend shelf life, or stabilize materials for transportation purposes.

[0041] Preparation method

[0042] Reference Figure 1 , which shows exemplary process flows that can be used with various non-limiting embodiments of the present invention, from sample collection to conditioned medium preparation to the use of such conditioned medium.

[0043] 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; culturing the hair follicles in a medium for a duration sufficient for cells derived from the hair follicles to secrete the one or more bioactive factors, thereby conditioning the medium; and collecting the conditioned medium.

[0044] According to embodiments of the present invention, hair follicles are obtained from a subject using non-invasive methods (e.g., plucking and follicular unit extraction). In more specific embodiments, the extracted hair follicles can be cultured immediately. In another embodiment, the extracted hair follicles can be transported to a desired facility in the transport composition of WO2020 / 024045A1. In yet another embodiment, the extracted hair follicles can be cryopreserved for different amounts of time in the cryopreservation composition of WO2020 / 024045A1 before thawing and culturing.

[0045] It is known that PRP-derived supplements are used directly as a safe and effective treatment for many clinical indications in addition to being used as an alternative to animal serum or serum-free supplements for culturing cells or tissues in vitro (see, for example, reference [2]). Thus, in more specific embodiments, the medium used to prepare the conditioned medium comprises PRP or a derivative of PRP (e.g., platelet lysate). In a preferred embodiment, the medium comprises from about 1% to about 30% w / w of platelet lysate, preferably from about 5% to about 25% w / w of platelet lysate, and more preferably about 20% w / w of platelet lysate.

[0046] The development of autologous conditioned medium therapy can ensure higher clinical safety as well as regulatory and commercial expectations for clinical products. Thus, in one specific embodiment, the PRP or a derivative of PRP (e.g., platelet lysate) is autologous for the hair follicles used to condition the medium. According to certain embodiments, whole blood is drawn from a subject and PRP is prepared by centrifuging for a suitable period of time under conditions standard in the art. In one embodiment, the PRP can be activated by adding chemicals such as, but not limited to, thrombin or calcium chloride to enhance the release of growth factors into the composition, forming a platelet lysate. In a preferred embodiment, the concentration of the PRP-activating chemicals (e.g., thrombin and / or calcium chloride) is from about 20 mM to about 30 mM. Alternatively, according to certain embodiments, the medium can contain supplements such as blood substitutes, human recombinant growth media, or other commercially available xeno-free nutrients.

[0047] According to certain embodiments, no animal serum or any other animal components are used in the preparation of the conditioned medium. In one embodiment, a number of human hair follicles are cultured in a substantially autologous, completely xeno-free medium.

[0048] According to certain embodiments, the medium for culturing hair follicles can include, but is not limited to, basal media known in the art of the present invention. The basal medium can be prepared synthetically or a commercially prepared medium can be used. Examples of commercially available media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), and Modified Dulbecco Medium (Isocel Modified Dulbecco Medium). In some embodiments, the medium contains Knock Out Dulbecco’s Modified Eagle Medium / F-12, MEM, BME, platelet lysate, or other xeno-free media known in the art.

[0049] According to certain embodiments, the medium can contain an effective amount of additional growth factors such as, but not limited to, basic fibroblast growth factor (b-FGF) to stimulate cell growth and expansion. In additional embodiments, the medium can also contain standard amounts in the art of additional antibiotics to prevent bacterial contamination, such as penicillin-streptomycin.

[0050] In one embodiment, the culture medium comprises components selected from the following: Knockout Dulbecco's Modified Eagle Medium / F-12, MEM, BME, or other xeno-free media known in the art, autologous platelet lysate at a concentration of about 5% to about 25% w / w, penicillin and / or streptomycin, or any combination thereof.

[0051] According to certain embodiments, hair follicles are cultured under optimal conditions to stimulate cell expansion. In some embodiments, the hair follicles are cultured at a temperature between about 20°C and about 50°C. In some preferred embodiments, the hair follicles are cultured at a temperature between about 30°C and about 40°C, and more preferably about 37°C.

[0052] According to certain embodiments, the hair follicles are maintained at a CO 2 percentage between about 2% and about 10%, preferably at about 5% CO 2 .

[0053] According to certain embodiments, the hair follicles can be cultured in the growth medium for a period of about 1 to about 6 weeks, preferably about 4 weeks. Refer to Figure 2 , which shows phase contrast microscopy images of follicle-derived cells growing out of the outer root sheath of hair follicles in culture on day 5 and day 12.

[0054] According to certain embodiments, during the culture phase of hair follicles in the growth medium, there is a heterogeneous cell population, which can include cells cultured according to standard procedures known to those skilled in the art, such as but not limited to keratinocytes, mesenchymal stem cells, fibroblasts, hair-related pluripotent cells, dermal papilla cells, and other primary somatic or stem cell types. For example, refer to Figure 3 , which shows immunofluorescence staining, which shows significant expression of the basal keratinocyte markers KRT14 and KRT5 in the expanded follicle-derived keratinocytes. Figure 4 Another exemplary cell type is shown in

[0055] which shows immunofluorescence staining of mesenchymal stem cells isolated from hair follicles for standard mesenchymal-related markers known in the art; the cells express the immunophenotypic profile typical of mesenchymal stem cells (CD90+CD105+CD73+CD44+ and CD45-CD31-). The culture of different cells and the expression of paracrine factors can affect the therapeutic efficacy of the conditioned medium described herein or compositions containing such conditioned medium.

[0056] According to certain embodiments, the conditioned medium is further processed. In one embodiment, the conditioned medium is centrifuged and filtered to purify the conditioned medium and remove cell 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 lyoprotectant (such as sucrose) is added to the conditioned medium as a protein stabilizer and to protect the conditioned medium during the lyophilization process; determining the optimal lyoprotectant and the effective amount of such a reagent is within the ordinary knowledge and skill of those in the art. In another embodiment, the lyophilized conditioned medium is suspended in a suspension mixture that comprises one or more of the following: saline, hyaluronic acid, silicone gel, petrolatum, PRP, derivatives of PRP, platelet lysate, and a pharmaceutically acceptable excipient.

[0057] According to a preferred embodiment, a method for preparing a conditioned medium comprising one or more bioactive factors comprises the following steps:

[0058] · Non-invasively extracting hair follicles from a subject by plucking or by follicular unit extraction;

[0059] · Culturing the hair follicles in a xeno-free medium or autologous PRP or a combination of both to induce cell expansion;

[0060] · Collecting the conditioned medium when the cultured cells reach confluence; and

[0061] · Optionally, further processing the conditioned medium with centrifugation, filtration, lyophilization, or other steps to ensure elimination of cellular material, purity, and stability of the final product.

[0062] Conditioned medium

[0063] According to certain embodiments, the present invention also provides a conditioned medium comprising one or more bioactive factors secreted by cells derived from hair follicles obtained from a subject. In some embodiments, such conditioned medium is prepared using the preparation methods described herein.

[0064] According to certain embodiments, one or more secreted factors may include regenerative factors, which may include proteins (such as growth factors, cytokines, chemokines), nucleic acids (such as miRNA), polysaccharides (such as hyaluronic acid), and / or combinations thereof, bound within extracellular vesicles or separate from extracellular vesicles (such as exosomes).

[0065] According to certain embodiments, one or more bioactive factors can 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, interleukin (e.g., IL-1, IL-2, IL-6), or any combination thereof, and their concentrations vary among subjects, ranging from 0 μg / mL to 1000 μg / mL. Reference Figure 5 , which shows an antibody-based cytokine array analysis showing the levels of several bioactive factors detected in a non-limiting embodiment of the conditioned medium.

[0066] According to certain embodiments, the present invention also provides a composition comprising the conditioned medium disclosed herein and one or more of the following: saline, hyaluronic acid, PRP, derivatives of PRP, platelet lysate, pharmaceutically acceptable excipients, carriers, and diluents. In another embodiment, the composition is formulated into a dosage form selected from injections, oily suspensions, hydrogels, nanogels, ointments, creams, serums, emulsions, sprays, patches, gels, drops, or any combination thereof. In a more specific embodiment, the present invention provides a pharmaceutical or cosmetic composition comprising conditioned medium, extracellular vesicles, autologous PRP, hyaluronic acid, or a combination thereof.

[0067] According to certain embodiments, the conditioned medium or composition is substantially free of animal serum or any animal components. Animal serum and other animal-derived components are widely used in cell and / or tissue culture as sources of growth factors, hormones, amino acids, lipids, and other nutrients. These are associated with biosafety issues and there are batch-to-batch variations. Although most prior art secretome therapies switch to serum-free media prior to collection, the use of animal products at any stage during cell culture remains a problem. When translated into clinical applications, introducing contaminants into the process and thus potentially into the final therapeutic product is a major safety concern.

[0068] According to certain embodiments, the conditioned medium or composition comprises additional soluble components, including but not limited to growth factors from the growth medium, which are intended to stimulate the growth and expansion of follicle-derived cells cultured therein. In one embodiment, such growth factors are derived from PRP or derivatives of PRP contained in the medium. In another embodiment, growth factors are added to the medium, including but not limited to from about 0.01 to about 10 ng / ml of recombinant human basic fibroblast growth factor, or other recombinant, synthetic or human-derived growth factors.

[0069] According to certain embodiments, the conditioned medium or composition is substantially free of intact cells or cell debris. In one embodiment, the conditioned medium is a cell-free secretome product prepared from a heterogeneous population of primary cells cultured from human hair follicles.

[0070] According to certain embodiments, the conditioned medium or composition is autologous.

[0071] According to a preferred embodiment, the conditioned medium or composition is autologous and xenogeneic-free, and is prepared by culturing human hair follicles under xenogeneic-free conditions to stimulate the growth of follicle-derived cells. In another preferred embodiment, the conditioned medium or composition is prepared by culturing human hair follicles in PRP or derivatives of PRP to stimulate the growth of follicle-derived cells.

[0072] According to certain embodiments, the conditioned medium or composition can be used for therapeutic or cosmetic purposes, including for wound healing, stimulating soft tissue or connective tissue repair / regeneration in a targeted tissue site (e.g., hair regeneration, skin regeneration, reducing or preventing signs of skin aging, or treating joint-related disorders), or delivering one or more bioactive factors to a target tissue site. See Figure 6 , which compared the wound healing of human skin cells cultured in standard growth medium with that of human skin cells cultured in standard growth medium supplemented with the conditioned medium of a preferred embodiment of the present invention by a scratch assay.

[0073] In one embodiment, a therapeutically effective amount of the conditioned medium or composition is administered to a subject or the subject's cells or tissues for a suitable period of time. In a preferred embodiment, the conditioned medium or composition is used for autotherapy of the subject from whom the hair follicles are collected. In another embodiment, the conditioned medium or composition can be used for allogeneic therapy of a subject, wherein the conditioned medium is prepared from the hair follicles of a healthy donor.

[0074] According to certain embodiments, the conditioned medium can be used fresh directly after preparation or lyophilized for storage. In one embodiment, the lyophilized conditioned medium can be stored at room temperature for about 12 weeks or at about 4 °C for a period of up to about 24 months. In another embodiment, the lyophilized conditioned medium can be mixed with a solid or liquid carrier, including PRP, hyaluronic acid, saline, or a cream or serum, for topical or injectable applications.

[0075] Kit

[0076] According to certain embodiments, the present invention provides a kit comprising a medium suitable for culturing hair follicles and a user's manual for preparing a follicle-derived conditioned medium for a subject.

[0077] Example

[0078] The present invention will be further illustrated by the following non-limiting examples. These examples are set forth to aid in understanding the disclosure but are not intended and should not be construed to limit the scope of the present invention in any way.

[0079] The examples do not include a detailed description of conventional methods well known and known to those skilled in the art.

[0080] Example 1 - Preparation of Conditioned Medium by Culturing Hair Follicles in Autologous Platelet Lysate

[0081] This example illustrates a preferred method for preparing a conditioned medium containing one or more bioactive factors using hair follicles according to one aspect of the present invention.

[0082] The hair follicles were thoroughly rinsed with an antibiotic-antifungal solution, cut to a position 1-2 mm distal to the outer root sheath, and then inoculated into a culture vessel pre-coated with CELLstart matrix. The hair follicles were then incubated at 37 °C for a period of two to four weeks with 5% CO 2 and 5% O 2 The culture growth medium consisting of 20% autologous platelet lysate diluted in a basal medium was added to the inoculated hair follicles. The culture growth medium was changed every two days to maintain cell viability.

[0083] Once the follicle-derived cells reached 70-90% confluence, the conditioned medium was collected, centrifuged at 300 x g for 5 minutes, and the supernatant was filter sterilized through a disposable sterile 0.22 µm filter system.

[0084] The resulting solution was then aliquoted into sterile vials and stored at 4 °C for immediate use or lyophilized to facilitate long-term storage.

[0085] ​

[0086] Example 2 - In vitro wound healing assay comparing follicle-derived conditioned medium and standard growth medium

[0087] This example describes an in vitro wound healing assay to show the effect of follicle-derived conditioned medium on wound healing compared to standard growth medium alone as a control.

[0088] Subject skin cells were cultured in a 12-well plate until confluent. Once confluent, a defect was created by scraping the cells from the plate surface using a scalpel. This resulted in the formation of an artificial wound where cells were missing due to the scraping operation.

[0089] The artificially wounded skin cells were then cultured in standard growth medium known to promote proliferation and migration, or in standard growth medium containing 10% conditioned medium prepared according to the method described in Example 1.

[0090] Wound closure, cell migration, and proliferation of the artificially wounded skin cells were observed and measured. As Figure 6 shown, compared to control skin cells cultured only in standard growth medium, skin cells cultured in 10% conditioned medium after the introduction of an artificial wound showed an approximately 3.5-fold increase in wound closure after approximately only 48 hours of culture through enhanced and combined cell proliferation and migration. The results of this wound healing assay show improved wound healing associated with follicle-derived conditioned medium.

[0091] Example 3 - Follicle-derived conditioned medium as a potential treatment for androgenetic alopecia

[0092] This example describes a clinical trial to evaluate the efficacy of follicle-derived conditioned medium for treating hair loss conditions such as androgenetic alopecia.

[0093] Subjects diagnosed with androgenetic alopecia were selected for this study. The following inclusion criteria were used:

[0094] · Male subjects with a clinical diagnosis of androgenetic alopecia.

[0095] · Subjects must consent to the following sample collections:

[0096] ο At least 20 follicles plucked from the posterior scalp, and

[0097] ο A total of 30 ml of blood.

[0098] Follicle and blood samples were collected from the subjects according to the preparation method described in Example 1 to allow for the preparation of autologous follicle-derived conditioned medium.

[0099] The resulting conditioned medium is lyophilized. The lyophilized conditioned medium is suspended in PRP, hyaluronic acid, saline, or any suitable solvent or carrier.

[0100] The subject is administered an effective dose of the conditioned medium locally or intradermally to the treatment site once a month for five months.

[0101] The following baseline examinations are performed throughout the study: Trichogram analysis for evaluating hair density and anagen induction, clinical photographs, and ultrasonography for evaluating dermal thickness and echogenicity.

[0102] Example 4 - Conditioned medium derived from hair follicles as a potential treatment for improving wound healing and inhibiting scar tissue.

[0103] This example describes a clinical trial to evaluate the efficacy of conditioned medium derived from hair follicles on improved and accelerated wound healing after elective plastic surgery.

[0104] Subjects scheduled to undergo abdominoplasty (tummy tuck) to remove excess skin and fat from the abdomen:

[0105] · Female subjects with a scheduled surgical date for abdominoplasty.

[0106] · The subject must consent to the following sample collection:

[0107] ο At least 20 hair follicles are plucked from the posterior scalp one month before surgery.

[0108] Hair follicles are collected from the subject according to the preparation method described in Example 1 to allow for the preparation of autologous hair follicle-derived conditioned medium.

[0109] The resulting conditioned medium is lyophilized. The lyophilized conditioned medium is suspended in hyaluronic acid.

[0110] An effective dose of the conditioned medium is administered to the subject once a day for 7 days, locally to one half of the abdominoplasty. Hyaluronic acid is administered to the subject once a day for 7 days, locally to the posterior half of the abdominoplasty. This provides a split-face study of the treatment and control sites for each subject.

[0111] The following baseline examinations are performed throughout the study: clinical photographs and ultrasonography to evaluate dermal thickness, topography, scar tissue formation, and healing time.

[0112] Those skilled in the art will understand that there are more possible alternative embodiments and modifications, and the above examples are merely illustrative of one or more embodiments. Therefore, the scope is limited only by the appended claims.

[0113] Explanation

[0114] It should be understood that, for the purposes of this application, the language “at least one of X, Y, and Z” or “one or more of X, Y, and Z” can be interpreted to mean only X, only Y, only Z, or any combination of two or more items X, Y, and Z (e.g., XYZ, XYY, YZ, ZZ).

[0115] In this application, a component can be described as “configured to” or “capable of” performing one or more functions. Generally, it should be understood that a component that is configured to or capable of performing a certain function means that the component is configured to or capable of performing that function, or is suitable for performing that function, or is adapted to perform that function, or is operable to perform that function, or otherwise has the ability to perform that function.

[0116] References in this application to “an embodiment”, “one embodiment”, “implementations”, “variations”, etc. indicate that the described embodiment, implementation, or variation may include a particular aspect, feature, structure, or characteristic, but not every embodiment, implementation, or variation must include that aspect, feature, structure, or characteristic. Moreover, such phrases may or may not refer to the same embodiment mentioned in other parts of the specification. Additionally, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, whether or not explicitly described, it is within the knowledge of those skilled in the art to combine such module, aspect, feature, structure, or characteristic with other embodiments operably or connectively. In other words, in different embodiments, any module, element, or feature can be combined with any other element or feature, unless there is an obvious or inherent incompatibility, or it is explicitly excluded.

[0117] It should also be noted that claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as a precedent for the use of exclusive terms (e.g., “solely”, “only”, etc.) related to the expression or “negative” limitation of claim elements. The terms “preferably”, “preferred”, “prefer”, “optionally”, “may”, and similar terms are used to indicate that the item, condition, or step mentioned is an optional (non-essential) feature of the present invention.

[0118] It should also be understood that when used herein, the terms “comprising”, “containing”, “including”, and / or “encompassing” specify the presence of the stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0119] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural references. The term "and / or" means any one of the items associated with that term, any combination of the items, or all of the items. The phrase "one or more" is readily understood by those skilled in the art, particularly when read in the context in which it is used.

[0120] As will be understood by those skilled in the art, for any and all purposes, particularly in providing a written description, all ranges recited herein also cover any and all possible sub-ranges and combinations of such sub-ranges, as well as the individual values that make up the range, particularly integer values. The recited ranges include each specific value, integer, fraction, or particular identity, etc., within the range. Any recited range can be readily identified as fully described and enabling that same range to be at least broken down into equal halves, thirds, quarters, fifths, or tenths. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc.

[0121] References

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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. A method for preparing a conditioned medium containing one or more biologically active factors, the method comprising: include: obtaining hair follicles, the hair follicles being non-invasively harvested from a subject; culturing the hair follicles in a culture medium for a duration sufficient to allow the hair follicle-derived cells to secrete the one or more bioactive factors, thereby conditioning the culture medium; and The conditioned medium was collected.

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

3. The method of claim 2, wherein the PRP, derivative of PRP, or platelet lysate is autologous to the hair follicles.

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. 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 growth factor or basic fibroblast growth factor, or any combination thereof.

6. The method of 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 a confluence of about 80% to about 90%.

8. The method of any one of claims 1 to 7, further comprising at least one of the following steps: centrifuging and filtering the conditioned medium to remove cell debris; cryopreserving, freezing or lyophilizing the conditioned medium; and The lyophilized conditioned medium is suspended in a suspension mixture comprising one or more of the following: saline, hyaluronic acid, silicone gel, petrolatum, PRP, a derivative of PRP, platelet lysate, and a pharmaceutically acceptable excipient. 9 . A conditioned medium prepared using the method according to claim 1 .

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

11. The conditioned medium of claim 9 or 10, wherein the one or more bioactive factors comprise growth factors, cytokines, chemokines, small peptides, nucleic acids, extracellular matrix molecules, extracellular vesicles, factors capable of mediating physiological processes, or any combination thereof.

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

13. The conditioned 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, interleukins (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. The conditioned medium of any one of claims 9 to 13, wherein the conditioned medium is substantially free of intact cells, cell debris and / or is xeno-free.

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

16. The composition of claim 15, wherein the PRP, derivative of PRP, or platelet lysate is autologous to the hair follicle-derived cells that condition the conditioned medium.

17. The composition of claim 15 or 16, formulated into a dosage form selected from the group consisting of injection, oily suspension, hydrogel, nanogel, ointment, cream, serum, emulsion, spray, patch, gel, drops, or any combination thereof.

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

19. The use of claim 18, wherein the therapeutic or cosmetic treatment comprises stimulating soft tissue or connective tissue repair in a targeted tissue site, hair regeneration, skin regeneration, reducing or preventing signs of skin aging, or treating joint-related conditions.

20. The use of claim 18 or 19, wherein the targeted tissue site is autologous to the hair follicle-derived cells that condition the conditioned medium.

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