Culture media containing amphibian bioactive molecules and uses thereof

Abstract

The present disclosure describes obtaining culture media including spent media from growing in vitro amphibian cells. The culture media contain bioactive molecules for treating skin conditions.

Description

Attorney Docket No. R217-0016PCTCULTURE MEDIA CONTAINING AMPHIBIAN BIOACTIVE MOLECULES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 745,201 , filed January 14, 2025, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The disclosure describes culture media used to grow amphibian cells and the use of the media for various purposes.BACKGROUND

[0003] Although all mammals including humans can spontaneously regenerate the tips of their fingers into adult life, mammals are not able to regenerate their limbs in contrast to amphibians. Most amphibians can regenerate missing body parts. However, their regenerative ability to regenerate varies extensively from species to species. Urodeles, for example, have exceptional regenerative capabilities.

[0004] Wound healing in amphibians and humans are similar. However, adult mammalian skin wound repair commonly results in scar tissue formation, while amphibians repair wounds by regeneration instead of scarring. It is desirable to develop methods for treating mammalian wounds that would not lead to scarring and in a shortened period.

[0005] A number of amphibian-derived wound healing molecules, including peptides have been reported. These molecules exist in skin secretions and contribute to skin repair in amphibians. Thus, there is an interest in developing ways to obtain the wound healing molecules of amphibians, in particular Urodeles, including those secreted by amphibians for treating mammalian skin.

[0006] There is also an interest in developing ways to easily obtain amphibian cells, especially Urodela cells, in large quantities and also the molecules that they secrete in large quantities. However, immortalized Urodela cell lines are difficult to establish (Satoh et al., Zoological Letters, 2022, 8:6, doi.org / 10.1186 / s40851 -022-00190-6).SUMMARY

[0007] This Summary is provided to introduce a selection of concepts in a simplified form that is further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0008] The present disclosure describes obtaining culture media used to grow amphibian cells for treating skin conditions and for growing other amphibian cells. In embodiments, the cultureAttorney Docket No. R217-0016PCTmedia can be collected when the in vitro amphibian cells reach confluency in a container. The culture media can also be collected 12 hours (hrs) to 72 hrs after growing the in vitro amphibian cells in a container. The culture media can also be collected as a spent media, after the amphibian cells have been starved -serum has been withdrawn or reduced from the culture media-for a period of time. The spent media can be collected 12 to 72 hrs after the amphibian cells have been grown in the culture media without serum (serum free media). The culture media can be further processed by concentration, filtration, or dialysis to remove undesirable agents from the culture media.BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG. 1 shows the different types of cells that are present in the in vitro amphibian cells prepared by the methods described herein after one passage.

[0010] FIGs. 2A-2D shows the doubling times of the in vitro amphibian cells. FIG. 2A shows the growth of a population of cells in an area of 8742 pixels (px) on Day 6 after explantation from an amphibian. FIG. 2B shows the same population of cells has grown to the size of 28364 px on Day 7. FIG. 2C shows that the same population of cells has grown to the size of 72836 px on Day 8. FIG. 2D shows that the same population of cells has grown to the size of 139588 px on Day 12.

[0011] FIG. 3 shows proteins are present in the spent media of in vitro axolotl cells. The spent media were collected 48 hours after culturing the axolotl cells without FBS. The proteins in the spent media were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). The SDS-PAGE shows that the spent media contain proteins having molecular weight ranging from about 12 kilodaltons (kD) to about 270 kD, with a majority of the proteins having molecular weight around 50 kD to 75 kD.DETAILED DESCRIPTION

[0012] The present disclosure describes growing in vitro amphibian cells and obtaining culture media used to grow the amphibian cells for treating skin conditions and for growing amphibian cells.

[0013] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0014] The term “in vitro amphibian cells” refers amphibian cells grown in an in vitro system including those that have been passaged one or more times and those from amphibian cell lines.

[0015] The term “antigen” refers to a molecule that is a toxin or is foreign to the subject and induces an immune response in the form of the production of antibodies against the molecule.Attorney Docket No. R217-0016PCTThe compositions or activated skin described herein can have reduced antigenicity as compared to native skin, such that it can be used in a subject.

[0016] The term “bioactivity” refers to biological effects. A substance is “bioactive” or has “bioactivity” includes a substance having a biological function. Bioactive molecules include proteins, peptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes, and other functional molecules secreted by amphibians, such as axolotls.

[0017] The term "biocompatible" refers to a product and its normal degradation products in vitro, ex vivo, or in vivo that are substantially non-toxic and non-carcinogenic to a cell, tissue, organ, organism, or subject within useful, practical, and / or acceptable tolerances. The term "cytocompatible" refers to a product that can sustain the viability and growth of a population of cells.

[0018] The term “biomaterial” refers to a material suitable for in vitro, ex vivo, or in vivo use. As an example of in vivo use, the biomaterial is suitable for administering to a subject in need thereof. The material can be synthetic or natural. The compositions and activated skin described herein are examples of biomaterial.

[0019] The term "carrier" or “excipient” refers to a substance added to a composition that does not affect the active compound in the composition. The carrier can be a diluent. The excipient can be a substance added to the composition to facilitate the administration of the composition.

[0020] The term “cosmetics” refers to products (excluding pure soap) intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance. Examples of cosmetic benefits can include improving the appearance of skin such as improving the appearance of wrinkling and fine lines, removing oil and excess sebum, reducing the appearance of skin blemishes, cleansing, and conditioning the skin, toning, and tightening the skin, soothing irritation, and refreshing and cooling the skin.

[0021] The terms “media,” “culture media,” “complete media” and “spent media” are used to refer to any in vitro cell culture media that can be used to grow in vitro amphibian cells including Urodele cells, such as axolotl cells. The terms “media” and “culture media” are used interchangeably. The terms “media” and “culture media” include complete media and spent media. The term “complete media” refers to culture media containing serum, such as bovine serum and fetal bovine serum (FBS). The term “spent media” refers to media that have been depleted of at least some nutrients by the amphibian cells, for example, the cells have consumed some of the nutrients, and the media contain bioactive molecules secreted by the cells. Spent media can also include media that do not contain serum (serum free) or have reduced serum as compared to a complete media. Removing or reducing serum from the media starves the cells and increases the cells secretion of bioactive molecules. In embodiments, the spent media are collected 30 minutes (mins) to 72 hours (hrs) after the cells are cultured with media without serumAttorney Docket No. R217-0016PCTor with reduced serum as compared to complete media (after starvation). Culture media containing reduced serum or no serum can have serum reduced by 25% to 100%, 30% to 99%, 40% to 98%, 50% to 97%, 60% to 96%, 70% to 95%, 80% to 95%, or 90% to 95%, as compared to the complete media. In embodiments, the spent media can also be collected when the cells reach about 20% to 100% confluency in the medium containing serum (without starvation).

[0022] The term “drugs,” “pharmaceuticals,” or “therapeutics” refers to articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease and articles (other than food) intended to affect the structure or any function of the body of man or other animals.

[0023] The term "derive", "derived," or "derives" refers to a product obtained from any stated source by any useful method. For example, an activated skin derived from an amphibian refers to an activated skin obtained from a member of the amphibian class.

[0024] The term “exogenous” refers to a product that originated outside of the organism, tissue, cell, organ, or subject. In contrast, the term “endogenous” refers to a product that originated from the organism, cell, tissue, organ, or subject.

[0025] The term “extracellular matrix” or “ECM” refers to a natural scaffolding having a three-dimensional structure including biomolecules and minerals that provide biochemical support to surrounding cells. The ECM includes structural and non-structural biomolecules, such as collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. The ECM can be obtained from various sources of tissues including the skin and non-cutaneous tissues.

[0026] The term "decellularized extracellular matrix" or "decellularized ECM" refers to ECM prepared by removing and / or devitalizing cells from ECM found in multicellular organisms, for example, amphibians or mammals. Decellularized ECM is substantially free of intact cells, lysed cells, and cellular components including cellular and nuclear debris such that the decellularized ECM exhibits reduced immunogenicity so that it can be administered to a subject, for example, a mammalian subject, as a non-toxic xenograft or biomaterial. A decellularized ECM that is “substantially free of immunogenic components” refers to an ECM in which immunogenic components are at a level that is not sufficient to induce an adverse immune response in a subject.

[0027] The term "isolated" refers to being separated or removed from its native surroundings, such that it is substantially free from components that accompany it in its naturally-occurring state. For example, a cell or a protein can be isolated from its naturally-occurring state.

[0028] The term “immunogenic” refers to relating to or producing an immune response. The term “immunogenicity” refers to the ability of a foreign substance, such as an antigen, to provoke an immune response in a subject. The compositions and activated skin described herein can have reduced immunogenicity as compared to the native skin, such that it can be used as a biomaterial in a subject.Attorney Docket No. R217-0016PCT

[0029] The term "non-toxic" refers to a product that causes little or no adverse reaction or substantial harm to cells and tissues in vitro or ex vivo, and / or does not cause a substantial adverse or undesirable reaction or substantial harm to cells and tissues in the body (in vivo).

[0030] The term “prevent” or “prevention” refers to the prevention of the onset, recurrence, or spread of a condition or one or more symptoms of the condition. As an example, the condition could be a skin condition. The term includes the administration of a product described herein before the onset of symptoms in particular to subjects at risk of developing a condition, such as a skin condition. The term includes the inhibition or reduction of one or more symptoms associated with the skin condition. The term “prevention” can be used interchangeably with the term “prophylactic treatment”.

[0031] The term “retain structural and functional integrity" used with reference to the ECM refers to retaining sufficient structure and function to permit and support the use of the matrix as a substrate for the growth of cells in vivo, ex vivo, or in vitro. For example, the decellularized ECM retains the structure and functional properties of a naturally occurring ECM enabling its use as a biomaterial.

[0032] The terms “scaffold” and “bioscaffold” are used interchangeably to refer to a substrate on which cells can grow in vitro, ex vivo, and / or in vivo. A scaffold or bioscaffold is an example of a biomaterial.

[0033] The terms “skin” and “skin sample” are used interchangeably to refer to the skin or a sample of skin from a subject.

[0034] The term “skin conditions” includes skin conditions that require therapeutic “drug” treatment including diseases, defects, and injuries including wounds, such as full-thickness wounds, deep dermal wounds, and burns.

[0035] The term “cosmetic skin conditions” includes skin conditions that are related to tone, clarity, radiance, brightness, elasticity, thinning, firmness, pore size, inflammation, and / or hydration of the skin.

[0036] The term "subject" refers to an animal, for example, a mammal. Examples of mammals include humans, dogs, cats, horses, cows, goats, sheep, pigs, and non-human primates. A subject in need of treatment or a subject in need thereof includes a subject having a disease or condition that needs to be treated. A subject in need thereof also includes a subject that needs treatment and / or prevention of a skin condition.

[0037] The term "therapeutically effective amount" refers to an amount of a product or composition that provides a therapeutic benefit in the treatment, prevention, or management of a condition or disease, such as a skin disease, an injury to the skin, or a wound, for example, a drug product. The term “therapeutically effective amount” also includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.Attorney Docket No. R217-0016PCT

[0038] The term "treatment" or "treating" in the context of administering a product, such as a biomaterial, to a subject refers to administering the product to achieve a desirable clinical / medical end-point, including alleviating symptoms of a disease or condition. Examples of such desirable end-points associated with skin disease or condition include wound healing, tissue closure, bulking tissue, preventing tissue adhesion, providing structural support to tissue, providing a protective barrier, and / or correcting a defect. Administering the product also includes applying the product to a subject.

[0039] The term “xenogenic” refers to a product derived or originated from a member of another species.

[0040] The term “amphibians” refers to cold-blooded vertebrate animals that include frogs, toads, newts, salamanders, and caecilians. They have an aquatic gill-breathing larval stage followed by a terrestrial lung-breathing adult stage. Amphibians include the class of amphibians and the orders of Anura (frogs or toads), Urodela (newts or salamanders), and Apoda (caecilians). In embodiments, the amphibians described herein are young or neotenic amphibians. A young amphibian includes a young frog, such as a froglet, tadpole, or larval stage young Apoda. The amphibian can be a young amphibian and the young amphibian can include a larval stage of any order of amphibian. The amphibian can be neotenic.

[0041] The term “salamanders” refers to a group of amphibians characterized by a lizard-like appearance and having a tail throughout life. The families of salamanders include the Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), Proteidae (mudpuppies and olms), Rhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The Ambystomatidae family includes Ambystoma altamirani, Ambystoma amblycephalum, Ambystoma andersoni, Ambystoma annulatum, Ambystoma barbourin, Ambystoma bishop, Ambystoma bombypellum, Ambystoma californiense, Ambystoma cingulated, Ambystoma dumerilii, Ambystoma flavipiperatum, Ambystoma gracile, Ambystoma granulosum, Ambystoma jeffersonianum, Ambystoma laterale, Ambystoma leorae, Ambystoma lermaense, Ambystoma mabeei, Ambystoma macrodactylum, Ambystoma maculatum, Ambystoma mavortium, Ambystoma mexicanum, Ambystoma opacum, Ambystoma ordinarium, Ambystoma rivulare, Ambystoma rosaceum, Ambystoma silvense, Ambystoma subsalsum, Ambystoma talpoideum, Ambystoma taylori, Ambystoma texanum, Ambystoma tigrinum, and Ambystoma velasci.

[0042] The families of salamanders are grouped under the order Urodela (or Caudata). The term “Urodele” refers to a salamander of the order Urodela, in the class Amphibia. The amphibians can be from the orders Urodela, Anura, and Apoda. Urodeles begin life as aquatic animals in a larval state, and some undergo metamorphosis from a juvenile form with gills to an adult, terrestrial, air-breathing form with lungs. During metamorphosis, a Urodele's physicalAttorney Docket No. R217-0016PCTfeatures are altered in preparation for life on land. These alterations include caudal fin resorption, thickening of the skin, the development of dermal glands, and resorption of gills. Sexual maturity also occurs during this time in most Urodeles. However, some families of Urodeles are "neotenic," which means that individuals of such families, even after reaching sexual maturity, retain their juvenile aquatic form throughout their lives. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of neotenic salamanders. Instead of becoming a terrestrial amphibian, an adult axolotl remains aquatic and gilled. However, under certain circumstances, an axolotl will undergo metamorphosis and transform into a terrestrial form.

[0043] Axolotls possess pigment cells, called chromatophores, that are responsible for their colors. The chromatophores of axolotls include melanophores containing eumelanin, xanthophores containing pteridines, and iridophores containing crystallized purines. Eumelanin is a black-brown pigment; pteridine is a yellow and reddish pigment; and crystallized purine is an iridescent white pigment. These pigments which are encoded by their respective genes provide the different phenotypes of axolotls, such as wild-type, golden albino, leucistic, and melanistic.

[0044] Axolotls have the ability to fully regenerate lost or damaged body parts including organs, limbs, and parts of the central nervous system, throughout their entire life. Axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scar-less processes. The axolotl wound healing process resembles the scar-free healing process of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than do the corresponding events in postnatal mammals.

[0045] Although the skin structure of amphibians is similar to mammals, Urodeles and anuran amphibians (frogs and toads) can regenerate their skin structures including the dermis and secretion glands without forming any scar after a deep skin injury. Moreover, the skin of amphibians contains ECM, which is rich in growth factors, which are favorable for wound healing. The ECM is a three-dimensional network of extracellular macromolecules and minerals including collagen, enzymes, glycoproteins, and hydroxyapatite which provide structural and biochemical support to surrounding cells. The ECM can include a combination of fibrous and network-type collagens. Examples of various types of collagens, such as one or more of type I, II, III, IV, V, and VI collagens. The ECM can also include elastin and / or elastic fibers. The ECM can also include laminin, fibronectin, hyaluronan, chondroitin sulfate, or both, and / or one or more proteoglycan, glycoprotein, glycosaminoglycan, or any combination thereof. The components and structure of the ECM play an important role in the healing process because the ECM components create scaffolding which provides the structural architecture of the matrix required for the healing process. Moreover, the ECM components are involved in stimulating the adhesion and migration of cells during the healing process as well as mediating the interactions among the cells andAttorney Docket No. R217-0016PCTbetween the cells and the matrix, or between ECM proteins during the healing process. Further, the ECM components also serve as a reservoir and modulator of the action of the cytokines and growth factors to regulate wound repair activities.

[0046] The present disclosure describes in vitro amphibian cells, methods of growing the in vitro amphibian cells, and methods of obtaining the culture media from growing the in vitro amphibian cells. Cells can be obtained from organs, tissues, and / or bodily fluids from amphibians, such as Urodeles and more specifically axolotls, and cultured using in vitro tissue cell culture systems. Examples of organs and tissues include skin, limb, and tail of an amphibian. Examples of bodily fluid include urine, blood, and saliva. Cells can also be obtained from reprogrammed amphibian cells that have been differentiated from one form of amphibian cells to another. The amphibian cells can also be cells that have been chemically or mechanically stimulated to become reprogrammed amphibian cells. The amphibian cells can also undergo natural mutation and lose their abilities to undergo apoptosis and become immortalized cells. The amphibian cells can be used for preparing in vitro amphibian cells including amphibian cell lines.

[0047] The in vitro amphibian cells are obtained by growing the amphibian cells in an in vitro cell culture system, which can include one or more containers for growing the cells, one or more cell culture media, one or more cell culture incubators, and / or one or more bioreactors. The container can be a flask such as a T25 to T175 flask. The containers used to grow the amphibian cells can be coated with collagen such as collagen such as collagen I, treated and untreated polystyrene, poly-amino acids, gelatin, ECM proteins, RGD-Motifs and fibronectin domains, Matrigel, alginate gels, and lactate gels.

[0048] The cell culture media used to grow in vitro amphibian cells include water, one or more carbon sources, one or more serums, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more polyphenols, one or more dipeptide supplement, one or more cell culture media supplements, or a combination thereof. Examples of carbon sources include glucose, fructose, sucrose, and other carbohydrates essential for cell growth and metabolism. Examples of nitrogen sources include ammonium salts, nitrates, urea, peptone, yeast extract, and amino acids. Examples of mineral salts include ferric nitrate, magnesium sulfate, and calcium chloride, Examples of serums include fetal bovine serum (FBS) and bovine calf serum (BCS). Examples of trace elements for cell culture include iron, copper, zinc, selenium, manganese, chromium, cobalt, molybdenum, and nickel. Examples of pH regulators include bicarbonate system such as sodium bicarbonate and CO2 incubator, and HEPES buffer. An example of osmolality regulators includes sodium chloride. Examples of steroid includes hydrocortisone, prednisolone, and dexamethasone. Examples of proteins includes a glycoprotein such as fibronectin, transferrin, insulin, and fetuin. Examples ofAttorney Docket No. R217-0016PCTamino acids include glutamine, glycine, asparagine, arginine, cysteine, tyrosine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Examples of dipeptide supplements include L-alanyl-L-glutamine, glycyl-L-glutamine, and tyrosine-alanine. Examples of antibiotics include penicillin, streptomycin, gentamicin, kanamycin, ampicillin, zeocin, amphotericin B, and Normocin. Examples of inhibitors can include ROCK and geraniin. Examples of reducing agent include beta-mercaptoethanol (BME), dithiothreitol (DTT), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP). An example of a polyphenol includes geraniin. Examples of cell culture media supplements (CCMS) such as insulin-transferrin-selenium (ITS-G) solution, insulin-transferrin-selenium-ethanolamine (ITS-X) solution, and insulin-transferrin-selenium-sodium pyruvate (ITS-A).

[0049] The culture media used to culture the amphibian cells can include a commercially available medium. Examples of commercially available cell culture media include Dulbecco’s Modified Eagle Medium (DMEM), Eagles Minimum Essential Medium (EMEM), Roswell Park Memorial Institute (RPMI) 1640, Iscove’s Modified Dulbecco’s Medium (IDM), Leibovitz Medium, and Glasgow’s Minimum Essential Medium (GMEM).

[0050] Supplements that are needed can be added to the commercially available cell medium. Examples of supplements include one or more serum, protein, trace element, antibiotics, steroids, proteins, and other described immediately above.

[0051] In embodiments, exemplary culture media can be prepared using the components listed in Table 1. The components listed in Table 1 for coating are used to coat the containers for growing the cells prior to adding the culture media or the cells.Table 1Attorney Docket No. R217-0016PCT

[0052] Examples of culture media include: (1) EMEM, FBS, and CCGW; (2) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, and CCGW; (3) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, and CCGW; (4) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITZ-X, and CCGW; (5) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-G, ROCK inhibitor, and CCGW; (6) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, GlutaMax®, and CCGW; (7) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, 1X ITS-X, Hydrocortisone, GlutaMax®, and CCGW; (8) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, GlutaMax®, Geraniin, BSA, 2-ME and CCGW; (9) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, GlutaMax®, Geraniin, BSA, 2-ME, HEPES, Puromycin, Blasticidin, Hygromycin, Neomycin, or Zeocin, and CCGW; (10) EMEM, FBS, Pen-Strep, Normocin, Amphotericin, ITS-X, Hydrocortisone, GlutaMax®, and CCGW; (11). EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-X, Hydrocortisone, GlutaMax®, Geraniin, BSA, 2-ME, and CCG; (12) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-X, Hydrocortisone, GlutaMax®, and CCGW; (13) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-X, Hydrocortisone, GlutaMax®, HEPES, and CCGW; and (14) EMEM, FBS, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, GlutaMax®, Geraniin, BSA, 2-ME, HEPES, and CCGW.

[0053] The containers for growing the amphibian cells are coated with a coating for 15 to 45 minutes (mins) prior to adding the cells. In embodiments, the coatings include Collage I (Rat Tail) coating or Geltrex coating.Attorney Docket No. R217-0016PCT

[0054] In embodiments, culture media and coatings for growing in vitro amphibian cells include those shown below.1 . EMEM 70%, 2% FBS, and remaining volume: cell culture grade water (CCGW).2. EMEM 70%, 2% FBS, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, and remaining volume CCGW.3. EMEM 70%, FBS 2%,1X Pen-Strep, 1X Normocin, 1X Amphotericin B, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) for 30 minutes (mins). 4. EMEM 70%, 2% FBS, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes5. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 10 pM ROCK inhibitor, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.6. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.7. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.8. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2 mM GlutaMax®, 10 pM Geraniin, 5 mg / mL BSA, 50 pM 2- mercaptoethanol (ME), and remaining volume CCGW; coat plate with 100 ug / mL Collagen I (Rat Tail) coating for 30 mins.9. EMEM 70%, FBS 0-2%, 0-1 X Pen-Strep, 0-1 X Normocin, 0-1 X Amphotericin B, 1X ITS- G, 0.33 pg / mL Hydrocortisone, 2 mM GlutaMax®, 0-10 pM Geraniin, 0-5 mg / mL BSA, 50 pM 2-ME, 2-5% HEPES, 0 - 10 mg / mL Puromycin, Blasticidin, Hygromycin, Neomycin, or Zeocin, and remaining volume CCGW; coat plate with Geltrex coating.10. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.11. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 10 pM Geraniin, 5 mg / mL BSA, 50 pM 2-ME, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.12. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.Attorney Docket No. R217-0016PCT13. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 1% HEPES, and remaining volume cell CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes. 14. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 10 pM Geraniin, 5mg / mL BSA, 50 pM 2-ME, 1% HEPES, and remaining volume CCGW; and coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes.

[0055] Culturing the amphibian cells include growing the cells in culture media, removing the culture media a couple of hours to 21 days post excision to collect the migrated cells, passaging the cells when the cells reach confluency in a container, such as a flask, and passaging the cells until the cells become a cell line which will grow or proliferate continuously.

[0056] Culturing the cells also include changing the culture media every 24 to 72 hours. Collecting the migrated cells can include centrifuging the media, removing the supernatant, dispersing the cell pellet with culture media, and placing the dispersed cell pellet in a new flask. Culturing the cells also include subculturing or passaging the cells until the cells are immortalized and become a cell line. In embodiments, the cells are passaged at least 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, or 50 times.

[0057] A cell line is a defined population of cells with a uniform genetic makeup (clonal) that can be maintained in vitro, can be subcultured, and will proliferate indefinitely given appropriate fresh medium and space to yield an inexhaustible source of material that retains stability of certain phenotypes and functions. The cell line can be prepared by screening the cultured in vitro cells for immortal cells and subculturing or passaging the individual immortal cells to yield clonal cell lines. A clonal cell line refers to a population of cells that are genetically identical and descended from the same ancestor cell. A clonal cell line provides a homogeneous population of cells.

[0058] Obtaining a clonal cell line includes selecting a single immortalized in vitro amphibian cell with the desired characteristics from a population of cells and growing the single cell to a homogeneous population of cells. In embodiments, a cell sorter such as a fluorescence-activated cell sorting (FACS) machine can be used for selecting a single cell with the desired robust characteristics. Immortalized cells are assessed based on various characteristics including proliferation rate or doubling time, morphology, cell type, function, genetic stability, and capacity to be manipulated. Other characteristics that can be assessed include the expression of certain gene markers such as telomerase reverse transcriptase (TERT) and telomerase-associated protein 1 (TEP1) and the lack of expression of beta-galactosidase over several passages. TERT and TEP1 are gene markers for cell proliferation. These genes are up regulated in cells, such as stem cells, that are rapidly dividing. TERT elongates the telomeres of cells which increases the lifespan of the cells by allowing for indefinite division without shortening of the telomeres. Betagalactosidase is a senescence marker as it is a lysosomal enzyme. During senescence, there isAttorney Docket No. R217-0016PCTan increased lysosomal activity. Therefore, increased beta-galactosidase activity measured at pH 6.0 indicates senescence, which is undesirable. These characteristics can be used to distinguish the desired cell from the rest of the in vitro amphibian cells. Western blot PCR, and biochemical assays can be used to detect activation of immortal cell markers or deactivation of senescent cell markers. Senescence cell killer (SSK1), chemical selective agent, can be used to kill senescent cells. Karyotyping or whole-genome sequencing can be used to show genome stability which indicates immortalization.

[0059] Single cell RNA sequencing (scRNA-seq) or RNA sequencing (RNA-seq) can be used to confirm transcriptomic uniformity for showing clonal cell line derivation. As the cells become a clonal cell line, after at least 20 to 30 passages, the doubling time of the cells starts to stabilize.

[0060] The cell identity and phenotype are confirmed by immunocytochemistry for KRT5, KRT14, p63, vimentin, FSP-1, E-cadherin. Flow cytometry can be used to quantify the percentages of each lineage. Transcriptomics can be performed using high-throughput technologies such as RNA-seq to obtain transcript information for comparing with corresponding known amphibian information. Western blot can be used to detect lineage markers. For example, p53 stops tumor growth (tumor suppression), so its loss can lead to accelerated growth. Also, screening for senescent and non-senescent cells.

[0061] In embodiments, the in vitro amphibian cells obtained by the methods described herein include squamous epithelial cells, keratinocytes, and fibroblasts after 1-5 passages as shown in FIG. 1. In embodiments, these in vitro amphibian cells obtained have rapid doubling time. As shown in FIGS. 2A-2D, the doubling time is about one day from Day 6 to Day 8 and from Day 8 to Day 12 the doubling time is about 4 days. In embodiments, the in vitro amphibian cells described herein are from a clonal cell line.

[0062] The present disclosure describes collected culture media from growing in vitro amphibian cells, such as Urodela cells, in particular, axolotl cells. The cells can be immortalized amphibian cells or amphibian cells that have not yet been immortalized. In embodiments, the culture media can be collected 5 minutes (mins) to 7 days, 1 hour (hr) to 6 days, 2 hrs to 5 days, 4 hrs to 4 days, 8 hrs to 2 days, 12 hrs to 48 hrs, 24 hrs to 48 hrs, 36 hrs to 48 hrs after growing the cells in the culture media, depending on whether the cells are starved or not by removing serum from the culture media. Culture media can be collected 5 mins, 10 mins, 15 mins, 20 mins, 25 mins to 29 mins after growing the cells with or without starvation. In embodiments, the culture media that the cells are grown in become spent media after the cells are grown in the media for at least 30 mins with starvation. In embodiments, the culture media that the cells are grown in become spent media after the cells reach at least 20% confluency without starvation. In embodiments, the spent media without starvation can be collected starting after 1 to 5 passages, when the in vitro amphibian cells reach about 20% confluency to 100% confluency in a container.Attorney Docket No. R217-0016PCTIn embodiments, the spent media without starvation can be collected when the cells reach 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% confluency.

[0063] In embodiments, the in vitro amphibian cells are cultured to a critical cell mass, for example 80% to complete confluency and the culture media can be changed to a serum-free, serum reduced, or serum protein-free culture media. The in vitro amphibian cells can be grown under conditions of serum starvation, such as growing in media that is either serum-free, serum reduced, or serum protein-free. Serum starvation can induce the secretion of more bioactive molecules and remove fetal bovine serum from the media. The cells are grown for 30 mins to 72 hours in serum free, serum reduced, or serum protein-free media before the media is removed and collected as spent media. The spent media can be collected from immortalized amphibian cells or cells that have not yet been immortalized.

[0064] After complete removal of serum or after reducing the serum in the culture media, the spent media used to grow the cells from the amphibian tissue or from a clonal cell line are collected. The spent media can be collected 30 mins to 72 hrs after starvation. In embodiments, the spent media, after starvation, are collected 1 hr to 72 hrs, 2 hrs to 72 hrs, 4 hrs to 72 hrs, 8 hrs to 60 hrs, 10 hrs to 50 hrs, 12 hrs to 50 hrs, 14 hrs to 50 hrs, 16 hrs 16 hrs to 48 hrs, 20 hrs to 48 hrs, or 24 to 48 hrs.

[0065] The collected media, cultured (with and without starvation) or spent media (with and without starvation), contain bioactive molecules from amphibians that are therapeutic and can be used to treat skin conditions. Examples of bioactive molecules include proteins, peptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes, and other functional molecules secreted by amphibians, such as axolotls.

[0066] As shown in Fig. 3, the spent media, collected at 48 hours after growth of in vitro axolotl cells without serum, contain proteins having molecular weight ranging from about 12 kD to about 270 kD. Most of the proteins have a molecular weight in the range of about 50 kD to 75 kD. Proteomics performed on the collected spent media confirmed that in vitro amphibian cells, such as axolotl cells, include bioactive proteins such as proteins and fragments thereof. Examples of the proteins found in the spent media of in vitro axolotl cells are shown in Table 1 below. The proteins found also include fragments of the proteins in Table 1 that are functionally active.Table 1. Proteins Found in the Spent Media of Axolotl Cells.Attorney Docket No. R217-0016PCT

[0067] The proteins in the spent media of amphibian cells are functionally related. They are involved in: structural / extracellular matrix (ECM) remodeling and wound closure; stress response, cytoprotection, and dedifferentiation; metabolic reprogramming (Warburg-like Shift); inflammatory modulation and cytokine signaling; developmental and morphogenetic regulators;Attorney Docket No. R217-0016PCTcytoskeletal and axonal remodeling; genomic integrity and repair; and neuro-sensory / signaling. Tables 2-9 shows examples of the proteins in each group.Table 2. Structural / ECM Remodeling & Wound ClosureTable 3. Stress Response, Cytoprotection, and DedifferentiationAttorney Docket No. R217-0016PCTTable 4. Metabolic Reprogramming (Warburg-like Shift)>Table 5. Inflammatory Modulation and Cytokine SignalingTable 6. Developmental and Morphogenetic RegulatorsAttorney Docket No. R217-0016PCTTable 7. Cytoskeletal and Axonal RemodelingTable 8. Genomic Integrity and RepairAttorney Docket No. R217-0016PCTTable 9. Neuro-Sensory / Signaling-Linked

[0068] The collected culture media and spent media can be used immediately or stored in the cold at 4 °C or frozen. The collected culture and spent media can be lyophilized (freeze dried) for storage. The collected culture media and spent media can be further processed by concentration, filtration, and / or dialysis to decrease the volume and / or to remove undesirable agents, such as antibiotics or toxins, and formulated for use or stored. Filtration can include membrane filtration and multimodal filtration. Examples of membrane filtration includes ultrafiltration, diafiltration, depth filtration, normal flow filtration and tangential flow filtration,

[0069] The present disclosure describes compositions including the culture media and spent media described herein. The compositions can include pharmaceutical or cosmetic composition. The pharmaceutical compositions can include one or more pharmaceutically acceptable carriers, and the cosmetic compositions can include one or more cosmetically acceptable carriers. The compositions can be formulated in the form of a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.

[0070] One or more agents can be added to the culture media including the spent media, or the compositions described herein. The one or more agents can be xenogenic to amphibians, for example a mammalian protein or a small molecule. In embodiments, the present disclosure provides a composition described herein and one or more agents that are xenogenic to amphibians. Examples of one or more agents xenogenic to amphibians include peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof. The proteins or peptides comprise growth factors, cytokines, or chemokines. The polymers comprise synthetic or natural polymers or copolymers. The drugs comprise retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics. The antibiotics comprise neomycin, polymyxin B, bacitracin, or a combination thereof. In embodiments, the one or more agents xenogenic to amphibians can be a mammalian molecule, for example, a mammalian peptide, protein, drug, or nutrient.Attorney Docket No. R217-0016PCT

[0071] In embodiments, the media and the compositions described herein containing the bioactive molecules can be used to treat and / or prevent various skin conditions including inflammatory skin conditions and injured skin having a wound or disrupted skin barrier. In embodiments, the culture media can be used to heal wounds.

[0072] Examples of inflammatory skin conditions include psoriasis; dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. Other skin conditions needing treatment include fine lines and / or wrinkles, aging, redness, abrasion, burns, cuts, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The burns include acute thermal burns including first, second, or third-degree burns.

[0073] The media and the compositions described herein can also be used in a skincare regimen for protecting the skin from damage including UV rays and environmental pollution and as an aesthetic agent for improving the appearance of the skin.

[0074] The media and compositions described herein can also be used in a skincare regimen for protecting the skin from damage including UV rays and environmental pollution and as an aesthetic agent for improving the appearance of the skin. The compositions described herein can be used to treat and improve photodamaged skin. Photodamaged skin is characterized by one or more skin changes including fine and coarse wrinkles, roughness, freckles, and pigmentation, such as brown spot, that occur as a result of prolonged exposure to the sun.

[0075] The media and compositions described herein can be used to treat or improve the appearance of uneven skin tones, visible hyperpigmentation, and dyschromia (changes in the color of the skin or nails). The compositions described herein can be used to treat and improve skin hydration, skin quality and texture, and reduce the appearance of fine lines and wrinkles.

[0076] The media and compositions described herein can be used to enhance treatment after non-ablative fractional laser. Facial non-ablative fractional resurfacing refers to the use of lasers to reduce the signs of aging, to improve skin texture, laxity (loss of skin elasticity), and tone, and to reduce the appearance of scars and stretch marks. The process involves the microscopic injury of the epidermal, the thin outer layer of the skin, and the superficial dermal layers of the skin, the vascular tissue that plays a key role in skin function that includes the steady supply of oxygenated blood and nutrients, for reducing the signs of photodamaged skin. Photodamaged skin results from premature aging of the skin due to exposure to sun’s ultraviolet (UV) radiation. The compositions described herein can increase healing after a non-ablative fractional laserAttorney Docket No. R217-0016PCTprocedure on the skin, including photodamaged skin and to decrease downtime (duration of discomfort).

[0077] The collected media can also be used as a media for growing in vitro amphibian cells.

[0078] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0079] Numbers expressing ranges or quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ± 20% of the stated value; ± 15% of the stated value; ± 10% of the stated value; ± 5% of the stated value; ± 4% of the stated value; ± 3% of the stated value; ± 2% of the stated value; ± 1% of the stated value; or ± any percentage between 1% and 20% of the stated value.

[0080] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, have, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise,” “comprises,” “have,” or “has” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. In embodiments, the lack of a material effect of a step is evidenced by the lack of a statistically-significant reduction in the process step in having a functional effect such as bioactivity. Lack of a material effect of an embodiment can include a lack of a statistically-significant improvement in obtaining a spent medium containing bioactive molecules or using the spent medium described herein in treating a skin condition obtaining a biological function or bioactivity. Lack of a material effect of a spent medium or composition comprising a spent medium can include lack of one or more bioactive molecules leading to lack of one or more bioactivities or biological functions.

[0081] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recitedAttorney Docket No. R217-0016PCTherein. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1 , 2, 2.5, 2.7, 3, 4, 5, 5.1 , 5.3, 5.8 and 6. Moreover, any ranges cited herein are inclusive.

[0082] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein.

[0083] The following examples and exemplary embodiments illustrate exemplary products, compositions, and methods provided herein. These examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure.EXEMPLARY EMBODIMENTS

[0084] The following are exemplary embodiments.1 . A culture medium obtained from growing in vitro amphibian cells, wherein the culture medium includes one or more amphibian bioactive molecules, and cell culture grade water (CCGW).2. The culture medium of embodiment 1 , wherein the culture medium includes spent medium or complete medium; optionally, wherein, the spent medium is collected after starvation and is serum free.3. The culture medium of embodiment 1 or 2, wherein the culture medium further includes one or more carbon sources, one or more serums, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements, one or more polyphenols, one or more dipeptide supplements, or a combination thereof.4. The culture medium of embodiment 1 or 2, wherein the culture medium is serum free and further includes one or more carbon sources, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements (CCMS), one or more phenols, one or more dipeptide supplements, or a combination thereof.Attorney Docket No. R217-0016PCT5. The culture medium of any one of embodiments 1 -4, wherein the culture medium includes Dulbecco’s Modified Eagle Medium (DMEM), Eagle’s Minimal Essential Medium (EMEM), Roswell Park Memorial Institute Medium (RPMI), Iscove’s Modified Dulbecco’s Medium (IMDM), Leibovitz medium, or Glasgow’s Minimum Essential Medium (GMEM).6. The culture medium of any one of embodiments 1 -5, wherein the culture medium includes one or more of EMEM, fetal bovine serum, Penicillin-Streptomycin (Pen-Strep), Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A.7. The culture medium of any one of embodiments 1 -6, wherein the culture medium is serum free and includes one or more of EMEM, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A. 8. The culture medium of any one of embodiments 1 -7, wherein the culture medium includes one or more of Puromycin, Blasticidin, Hygromycin, Neomycin or Zeocin.9. The culture medium of any one of embodiments 1 -8, wherein the amphibian cells are from a frog, toad, newt, or salamander.10. The culture medium of one of embodiments 1-9, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian includes a froglet, tadpole, Urodele, or larval stage young Apoda.11. The culture medium any one of embodiments 1-10, wherein the amphibian is a neotenic Urodele.12. The culture medium of any one of embodiments 1-11, wherein the amphibian is an axolotl.13. The culture medium of claim 1-12, wherein the one or more bioactive molecules include one or more proteins.14. The culture medium of claim 1-13, wherein one or more proteins include proteins whose function include: structural and / or ECM remodeling; wound closure; stress response, cytoprotection, and / or dedifferentiation; metabolic reprogramming; inflammation modulation and / or cytokine signaling; developmental and / or morphogenic regulation; cytoskeletal and / or axonal remodeling; or a combination thereof.15. The culture medium of claim 1-14, wherein the one or more bioactive molecules include keratin 5, collagen type 1 a2, periplakin, thrombospondin-1, thrombospondin-4, prominin-1, growth / differentiation factor 5, heat shock protein 70, histone H4, de-etiolated-like protein 1 , L-lactate dehydrogenase A, triosephosphate isomerase, GAPDH, G6PD, suppressor of cytokine signaling 5, MHC class I heavy chain, prolactin, nodal 2, thyroid hormone receptor a, developmental orphan receptor-1 , E2F1 , musashi-like 2L, and dynein axonemal heavy chain3, microtuble associated rpotein 215, tropomyosin, or a combination thereof.Attorney Docket No. R217-0016PCT16. A composition including the culture medium of any one of embodiments 1-15, and optionally, the composition includes one or more carriers.17. The composition of embodiment 16, wherein the composition is a pharmaceutical composition, and optionally including one or more pharmaceutically acceptable carrier; or wherein the composition is a cosmetic composition, and optionally including one or more cosmetically acceptable carriers.18. The composition of embodiment 16 or 17, wherein the composition further includes one or more agents xenogenic to amphibians; and optionally, wherein the one or more agents include one or more peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof.19. The composition of any one of embodiments 16-18, wherein the composition is formulated as a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.20. A method of obtaining culture medium of any one of embodiments 1 -15 including obtaining a population of cells from an amphibian, growing the cells in an in vitro cell culture system and collecting culture medium after growing the amphibian cells in the vitro cell culture system for 12 to 72 hrs; and optionally storing the collected culture medium.21 . The method of embodiment 20, wherein the method includes growing the cells in complete medium, changing from complete medium to serum free medium, growing the cells in the serum free medium for 12 to 72 hrs, and collecting the spent medium.22. The method of embodiment 20 or 21 , wherein the method further includes filtering, concentrating, and / or dialysis to obtain culture medium free of undesirable agents.23. The method of any one of embodiments 20-22, wherein the method further includes formulating the obtained culture medium as a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.24. The method of embodiment 20-23, wherein the in vitro cell culture system includes one or more of a container for growing the cells, cell culture medium, cell culture incubator, and / or a bioreactor.25. The method of any one of embodiments 20-24, wherein growing the cells in an in vitro culture system includes expanding the cells in culture medium and optionally, passaging the cells.26. The method of any one of embodiments 20-25, wherein culture medium includes:complete medium including one or more carbon sources, one or more serums, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture medium supplements, one or more polyphenols, one or more dipeptide supplements, or a combination thereof; orAttorney Docket No. R217-0016PCTserum free medium including one or more carbon sources, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture medium supplements (CCMS), one or more phenols, one or more dipeptide supplements, or a combination thereof.27. The method of any one of embodiments 20-26, wherein the culture medium includes complete medium including one or more of EMEM, fetal bovine serum, Penicillin- Streptomycin (Pen-Strep), Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A; or serum free medium including one or more of EMEM, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A.28. The method of any one of embodiments 20-27, wherein growing the cells includes growing them in an incubator at 20°C to 30°C, 20°C to 28°C, 22°C to 28°C, 24°C to 28°C, 25°C to 27°C, or 26°C.29. The method of any one of embodiments 20-28, wherein growing the cells includes growing them in an incubator with a carbon dioxide level at 1 % to 5%, 1 % to 4%, 1 % to 3%, or 2%. 30. A method of treating and / or preventing a skin condition including administering the culture medium of any one of embodiments 1 -15 or the composition of any one of embodiments 16-19 to the skin of a subject in need of treatment.31 . The method of embodiment 30, wherein the skin condition is an inflammatory skin condition; and optionally, wherein the inflammatory skin condition is psoriasis, dermatitis, acne, rosacea, or burn.32. The method of embodiment 30, wherein the method includes treating and / or preventing the skin from photodamage and environmental pollution.33. The method of embodiment 30, wherein the method includes treating and / or preventing uneven skin tones, visible hyperpigmentation, and dyschromia.34. The method of embodiment 30, wherein the method improves skin hydration, skin quality and texture, and reduce the appearance of fine lines and wrinkles, as compared to a control substance that do not contain the culture medium or the composition.35. The method of embodiment 30, wherein the method is used to treat subject after treatment with non-ablative fractional laser.Attorney Docket No. R217-0016PCTEXAMPLES

[0085] Example 1. Preparation of Exemplary Culture Media for Growing In Vitro Amphibian Cells

[0086] The ingredients listed for each exemplary cell culture medium are mixed in a container and used to growing in vitro amphibian cells, such as axolotl cells. The components listed as coating are used to coat the containers for growing the cells prior to adding the prepared culture media or the cells.1 . EMEM 70%, 2% FBS, and remaining volume: cell culture grade water (CCGW).2. EMEM 70%, 2% FBS, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, and remaining volume CCGW.3. EMEM 70%, FBS 2%,1X Pen-Strep, 1X Normocin, 1X Amphotericin B, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) for 30 minutes (mins). 4. EMEM 70%, 2% FBS, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes5. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 10 pM ROCK inhibitor, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.6. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.7. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.8. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2 mM GlutaMax®, 10 pM Geraniin, 5 mg / mL BSA, 50 pM 2- mercaptoethanol (ME), and remaining volume CCGW; coat plate with 100 ug / mL Collagen I (Rat Tail) coating for 30 mins.9. EMEM 70%, FBS 0-2%, 0-1 X Pen-Strep, 0-1 X Normocin, 0-1 X Amphotericin B, 1X ITS- G, 0.33 pg / mL Hydrocortisone, 2 mM GlutaMax®, 0-10 pM Geraniin, 0-5 mg / mL BSA, 50 pM 2-ME, 2-5% HEPES, 0 - 10 mg / mL Puromycin, Blasticidin, Hygromycin, Neomycin, or Zeocin, and remaining volume CCGW; coat plate with Geltrex coating.10. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.11. EMEM 70%, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 10 pM Geraniin, 5 mg / mL BSA, 50 pM 2-ME,Attorney Docket No. R217-0016PCTand remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.12. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, and remaining volume CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 mins.13. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-X, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 1% HEPES, and remaining volume cell CCGW; coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes. 14. EMEM 70% phenol free, FBS 2%, 1X Pen-Strep, 1X Normocin, 1X Amphotericin B, 1X ITS-G, 0.33 pg / mL Hydrocortisone, 2mM GlutaMax®, 10 pM Geraniin, 5mg / mL BSA, 50 pM 2-ME, 1% HEPES, and remaining volume CCGW; and coat plate with 100 pg / mL Collagen I (Rat Tail) coating for 30 minutes.

[0087] Example 2. Culturing Axolotl Cells and Collecting Culture Media

[0088] Tissues from an axolotl were excised and processed for explanting in a flask containing culture medium. The tissue was washed and treated with phosphate buffered saline (PBS) containing normocin, amphotericin B, and penicillin-streptomycin. The tissue was minced into small pieces and placed into a flask, such as a T25 to T175 flask coated with collagen I and containing culture medium. The flask was placed in an incubator at 26 °C and 2% CO2. The flask was checked daily for migration of cells. Once the cells have migrated, the pieces of tissue were removed. The media was changed every 48 hours.

[0089] At various timepoints post excision, for example a couple of hours and up to 21 days post excision, all the tissues were removed from the plate and the culture media were completely replaced with new media. Some of cells migrated into the culture media, while others attached to the flask were detached using trypsin-EDTA and culture media. The culture media containing the migrated and detached cells were collected. The collected culture media containing the migrated and detached cells were centrifuged at 300 x g for 5 minutes at room temperature (RT). The supernatant was removed, and the pellet was gently tapped to disperse the cells. Culture media were added to the cell pellet to evenly disperse the cell and added back to a new flask. The new flask was placed back into the incubator for growth daily.

[0090] Once the cells reached confluency in a flask, then the cells were passaged by splitting into T150 coated container. To split the cells, the culture media were removed and collected and the flask was rinsed with sterile PBS and removed. T rypsin-EDTA was added to cover the bottom of the flask, and the flask was incubated at 26 °C and 2% CO2for about 5 to 10 minutes and checked for detachment. Once completely detached, the complete media (containing Fetal Bovine Serum) were added to the flask to neutralize the Trypsin-EDTA solution. The complete media were then aliquoted into a tube for centrifugation for 5 minutes at 300 x g at RT. The cells were rinsed with sterile PBS and centrifuged 5 minutes at 300 x g at RT. The supernatant wasAttorney Docket No. R217-0016PCTremoved and the cell pellet is gently tapped to disperse the cells. The dispersed cells were suspended in complete media and aliquoted into two coated T150 flasks and incubated at 26 °C and 2% CO2. The cells were checked daily for growth. Culture media were collected before the cells are split and / or every 24 to 72 hours. The cells are passaged until they become a continuous cell line.

[0091] The culture media, which is also the spent media, were collected from growing before the cells are split and / or every 24 to 72 hours. The spent media were collected starting at about 3 to 5 passages and thereafter, when the cells reach about 80% to about 100% confluency.

[0092] The cells are assessed for various characteristics. As shown in FIG. 1 , the in vitro cells include squamous epithelial cells, keratinocytes, and fibroblasts after one passage. As shown in FIGS. 2A-2D, the doubling time is about one day from Day 6 to Day 8 and from Day 8 to Day 12 the doubling time is about 4 days.

[0093] To obtain a clonal cell line, a single cell with the desired characteristics is selected and subcultured. After each passage, the cells are sorted using FACS and the cells with the desired characteristics are selected and subcultured. The cells are screened for features such as proliferation rate or doubling time, morphology, cell type, function, genetic stability, and capacity to be manipulated using well-known methods. They are also screened for expression of certain gene markers, for example, telomerase reverse transcriptase (TERT), and telomerase-associated protein 1 (TEP1), and the lack of expression of beta-galactosidase over several passages. Western blot PCR, and biochemical assays can be used to detect activation of immortal cell markers or deactivation of senescent cell markers. Senescence cell killer (SSK1), chemical selective agent, can be used to kill senescent cells. Karyotyping or whole-genome sequencing can be used to show genome stability which indicates immortalization. The cells are selected, screened, and passaged until a select clonal cell exhibit immortal characteristics.

[0094] Example 3. Enhancing Secretion of Bioactive Molecules into the Spent Media and Collecting Spent Media

[0095] Once the axolotl cells reach a critical cell mass, the cells attached to the flask were washed twice with serum free media. The non-adherent cells that were in the media were removed with the media and pelletized. They were washed with serum free media and reintroduced into the same flask with the attached cells to maximize cell mass. The cells in the flask were cultured with serum free media for 24 to 72 hrs before the serum free media (spent media) containing the bioactive molecules were collected and further processed for analysis and use.

[0096] A sample of the collected spent media were processed for SDS-PAGE analysis. The SDS-PAGE confirms that the collected spent media contain proteins (Fig. 3). Proteomics were performed on the collected spent media. Table 1 shows the various proteins found in the sample of collected spent media.Attorney Docket No. R217-0016PCT

[0097] Example 4. Use of the Collected Culture and Spent Media

[0098] The culture and spent media collected from the in vitro cells were further processed by filtering or dialysis to remove undesirable agents such as antibiotics. The protein collected was about 30 pg / mL.

[0099] The collected culture and spent media are formulated for treatment of subjects in need thereof. The formulation is applied directly to the skin of a subject in need thereof.

[0100] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.

[0101] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.REFERENCESSatoh et al., Zoological Letters, 2022, 8:6, doi.org / 10.1186 / s40851 -022-00190-6

Claims

1. Attorney Docket No. R217-0016PCTCLAIMS1 . A culture medium obtained from growing in vitro amphibian cells, wherein the culture medium comprises one or more amphibian bioactive molecules, and cell culture grade water (CCGW).

2. The culture medium of claim 1 , wherein the culture medium is spent medium; and optionally, the spent medium is without serum.

3. The culture medium of claim 1 , wherein the culture medium further comprises one or more carbon sources, one or more serums, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements, one or more polyphenols, one or more dipeptide supplements, or a combination thereof; or wherein the culture medium is serum free and further comprises one or more carbon sources, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements (CCMS), one or more phenols, one or more dipeptide supplements, or a combination thereof.

4. The culture medium of claim 1 , wherein the culture medium comprises Dulbecco’s Modified Eagle Medium (DMEM), Eagle’s Minimal Essential Medium (EMEM), Roswell Park Memorial Institute Medium (RPMI), Iscove’s Modified Dulbecco’s Medium (IMDM), Leibovitz medium, or Glasgow’s Minimum Essential Medium (GMEM).

5. The culture medium of claim 4, wherein the culture medium comprises one or more of EMEM, fetal bovine serum, Penicillin-Streptomycin (Pen-Strep), Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A; or wherein the culture medium is serum free and comprises one or more of EMEM, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A.

6. The culture medium of claim 1 , wherein the amphibian cells are from a frog, toad, newt, or salamander.

7. The culture medium of claim 1 , wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian comprises a froglet, tadpole, Urodele, or larval stage young Apoda.

8. The culture medium of claim 1 , wherein the amphibian is a neotenic Urodele.Attorney Docket No. R217-0016PCT9. The culture medium of claim 1 , wherein the amphibian is an axolotl.

10. The culture medium of claim 1, wherein the one or more bioactive molecules comprise one or more proteins.

11. The culture medium of claim 1 , wherein one or more proteins comprise proteins whose function include: structural and / or ECM remodeling; wound closure; stress response, cytoprotection, and / or dedifferentiation; metabolic reprogramming; inflammation modulation and / or cytokine signaling; developmental and / or morphogenic regulation; cytoskeletal and / or axonal remodeling; or a combination thereof.

12. The culture medium of claim 1, wherein the one or more bioactive molecules comprise keratin 5, collagen type 1 a2, periplakin, thrombospondin-1, thrombospondin-4, prominin-1, growth / differentiation factor 5, heat shock protein 70, histone H4, de-etiolated-like protein 1 , L-lactate dehydrogenase A, triosephosphate isomerase, GAPDH, G6PD, suppressor of cytokine signaling 5, MHC class I heavy chain, prolactin, nodal 2, thyroid hormone receptor a, developmental orphan receptor-1 , E2F1 , musashi-like 2L, and dynein axonemal heavy chains, microtuble associated rpotein 215, tropomyosin, or a combination thereof.

13. A composition comprising the culture medium of any one of claims 1-12, and optionally, the composition comprises one or more carriers.

14. The composition of claim 13, wherein the composition is a pharmaceutical composition, and optionally comprising one or more pharmaceutically acceptable carrier; or wherein the composition is a cosmetic composition, and optionally comprising one or more cosmetically acceptable carriers.

15. The composition of claim 13, wherein the composition further comprises one or more agents xenogenic to amphibians; and optionally, wherein the one or more agents comprise one or more peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof.

16. The composition of any one of claims 13, wherein the composition is formulated as a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.

17. A method of obtaining culture medium of any one of claims 1-12 comprising obtaining a population of cells from an amphibian, growing the cells in an in vitro cell culture system and collecting the culture medium after growing the amphibian cells in the vitro cell culture system for 12 to 72 hrs; and optionally storing the collected culture medium.

18. The method of claim 17, wherein the method comprises growing the cells in complete medium, changing from complete medium to serum free medium, growing the cells in the serum free medium for 12 to 72 hrs, and collecting spent medium.

19. The method of claim 17, wherein the method further comprises filtering, concentrating, and / or dialysis to obtain culture medium free of undesirable agents.Attorney Docket No. R217-0016PCT20. The method of claim 17, wherein the method further comprises formulating the obtained culture medium as a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.21 . The method of claim 17, wherein the in vitro cell culture system comprises one or more of a container for growing the cells, cell culture media, cell culture incubator, and / or a bioreactor.

22. The method of claim 17, wherein growing the cells in an in vitro culture system comprises expanding the cells in culture medium and optionally, passaging the cells.

23. The method of claim 17, wherein culture medium comprises:complete medium comprising one or more carbon sources, one or more serums, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements, one or more polyphenols, one or more dipeptide supplements, or a combination thereof; orserum free medium comprising one or more carbon sources, one or more nitrogen sources, one or more mineral salts, one or more trace elements, one or more regulators of pH and / or osmolality, one or more growth factors, one or more antibiotics, one or more steroids, one or more proteins, one or more amino acids, one or more Rho-kinase (ROCK) inhibitors, one or more reducing agents, one or more cell culture media supplements (CCMS), one or more phenols, one or more dipeptide supplements, or a combination thereof.

24. The method of claim 17, wherein the culture medium comprisescomplete medium comprising one or more of EMEM, fetal bovine serum, Penicillin-Streptomycin (Pen-Strep), Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A; or serum free medium comprising one or more of EMEM, Pen-Strep, Normocin, Amphotericin B, ITS-G, Hydrocortisone, L-Alanyl-L-Glutamine Dipeptide, Geraniin, BSA, 2-Mercaptoethanol (2-ME), HEPES, and CCGW; and optionally, wherein the ITS-G is substituted with ITS-X and ITS-A.

25. The method of claim 17, wherein growing the cells comprises growing them in an incubator at 20°C to 30°C, 20°C to 28°C, 22°C to 28°C, 24°C to 28°C, 25°C to 27°C, or 26°C.

26. The method of claim 17, wherein growing the cells comprises growing them in an incubator with a carbon dioxide level at 1% to 5%, 1% to 4%, 1% to 3%, or 2%.

27. A method of treating and / or preventing a skin condition comprising administering the culture medium of any one of claims 1 -12 to the skin of a subject in need of treatment.

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