Production method of mammalian macrophage cell derived biotechnological product

Abstract

The invention relates to a method for producing biotechnological products derived within mammalian cells that can be used in anti-aging, wound healing, skin rejuvenating, beautifying or skin restoring creams, gels, sprays or serum products falling under the cosmetic sector. More particularly, the invention is related to a method for trans forming the media obtained when growing mammalian macrophage cells into a biotechnological product for the production of a natural, organic, biocompatible and highly effective value-added product without the addition of any chemical agents or preservatives other than biocompatible solutions.

Description

[0001] PRODUCTION METHOD OF MAMMALIAN MACROPHAGE CELL DERIVED BIOTECHNOLOGICAL PRODUCT

[0002] Field of the Invention

[0003] The invention is related to a method for the production of a natural , organic, biocompatible and highly ef fective value-added product derived from mammalian cells that can be used in antiaging, wound healing, skin rej uvenating, beauti fying or skin restoring creams , gels , sprays or serum products in the cosmetic sector and without the addition of any chemical agents or preservatives other than biocompatible solutions .

[0004] Background of the Invention

[0005] It is known that as a result of the loss of collagen and elastic proteins in the dermal layers and the deterioration of elasticity and skin thickness over time , fine lines and wrinkles occur on the skin and this is seen as one of the signs of aging in humans . In the art , surgical treatments such as faceli ft , laser surgery and skin peeling and interventions with inj ection treatments ( e . g . , BOTOX®) are known to prevent and / or delay skin disorders due to said aging .

[0006] Furthermore , when the skin is damaged, for example , cut , crushed, exposed to chemicals , or deprived of oxygen, regardless of the age of the subj ect , cell death occurs in the relevant region, whereas the body initiates a sequence of chemical signals that stimulate responses aimed at healing the af fected tissues . It is well-known that the body, in response to certain situations , releases collagen to help repair damaged tissue , among other reactions . In the technical field, there are commercial products that are released for aesthetic purposes and designed to reduce , prevent or slow wrinkles on the skin, and medical products that are designed to treat tissue damage and cell loss resulting from traumatic conditions .

[0007] Hyaluronic acid, collagen, or di f ferent vitamins obtained by either chemical agents or extraction methods are used in cream, gel , serum, or spray formulations known in the art . Chemical active ingredients cause problems in terms of biocompatibility, while other extraction-based biological products have deficiencies in terms of ef fectiveness .

[0008] The aim of the ongoing studies is to develop products that are non-allergenic, non-toxic, more biocompatible , highly biologically ef ficient , minimi ze chemical usage , and have a minimal impact on the environment .

[0009] Products derived from conditioned medium grown in various cell culture media and in which various growth factors are involved, and their use are known in the art .

[0010] The application titled " Skin Cream" filed as US2011294731A1 on 12 . 02 . 2010 may be referred to as an example of the current state of the art . Said document is a patent file relating to a skin conditioner comprising a compos ition comprising anti-aging cosmetics for topical application, and more particularly, a cell culture medium conditioned by cells grown in two-dimensional culture . The skin cream of the said patent comprises a conditioned medium produced by incubating a medium with substantially homogeneous eukaryotic cells , at least one growth factor or extract or concentrate is present in said conditioned medium, thereby providing treatment and / or prevention of skin defect . Another example of the current state of the art is the document titled "Cosmetic Composition For Improving Condition Of Skin, Containing Cell Culture Medium, Epidermal Growth Factor and Bovine Serum Albumin" filed on 17 . 11 . 2015 with the number W02016080732A1 . The mentioned document is a patent file on a cosmetic composition containing cell culture medium, epidermal growth factor and bovine serum albumin to improve the condition of the skin . The said document relates in particular to a cosmetic composition for improving the condition of the skin containing a cell culture medium, an epidermal growth factor and bovine serum albumin as active ingredients , to a method for improving the condition of the skin using the said product , and to a method for preventing or treating skin diseases . It is mentioned in the said document that the cosmetic composition can exhibit wrinkle-reducing and wound-healing ef fects with a small amount of EGF and therefore can be useful in improving the condition of the skin or in preventing or treating skin diseases .

[0011] The following files may also be referred to as an example of the background of the invention : the document titled "Medicinal Composition Comprising Ips Cell Culture Supernatant And Method For Producing Same , Cosmetic And Method For Producing Same , Antiaging Compos ition, Method For Inhibiting Disease Onset , Method For Treating Disease , Method For Treating Tissue Abnormality And Beauti fication Method" filed on 12 . 01 . 2016 with the application number WO2016111377A1 and "Cosmetic And Facial Regeneration Composition Derived From Potentiated Adipose Derived Cells And Supernatants Thereof" filed on 14 . 12 . 2016 with the application number US2017165194A1 .

[0012] As an alternative to the products mentioned in the above-listed documents , a natural , organic , biocompatible and highly ef fective biotechnological product , which can be used in antiaging, wound healing, skin rej uvenating or beauti fying creams , gels , sprays or serum products and production method thereof have been developed without the addition of chemical agents or preservatives .

[0013] Detailed Description of the Invention

[0014] The invention is related to a method for trans forming the media obtained when growing mammalian macrophage cells into a biotechnological product for the production of a natural , organic, biocompatible and highly ef fective value-added product without the addition of any chemical agents or preservatives other than biocompatible solutions . The biotechnological product obtained by the production method of the invention is also within the protection scope of the invention .

[0015] An obj ect of the invention is to provide a production method for a biotechnological product that allows for the elimination of the disadvantages of products that can be produced by adding various chemicals to the prior art .

[0016] The invention, in its most general form, is a method of manufacturing a biotechnological product for dermocosmetic purposes , comprising the following process steps : a ) preparation of the media, b ) growing mammalian cells until confluency, c ) collection of confluent media at separate time points , d) obtaining the final product by mixing the collected media with a material containing at least one of the group of cream, gel , spray, serum base in crude and / or lyophili zed form .

[0017] In an embodiment of the invention, 10% fetal bovine serum is used to form broths in the process step a . In an embodiment of the invention, an RPMI medium containing 1 % penicillin- streptomycin is used in the process step a .

[0018] In an embodiment of the invention, mammalian cells are grown at a concentration of lx! 06 / mL in the process step b .

[0019] In an embodiment of the invention, mammalian cells are grown until 90% confluent in the process step b .

[0020] In an embodiment of the invention, the media are collected in the process step c at hours 24 , 48 and / or 72 .

[0021] In one embodiment of the invention, the media collected in the process step d are mixed with a material containing at least one of the group of cream, gel , spray, serum base in 5% , 10% and / or 20% by volume .

[0022] In more detail , the method according to an embodiment of the invention comprises the following process steps ; a ) preparation of the media using RPMI media containing 10% FBS , 1 % Pen Strep, b ) growing mammalian cells until 90% confluency at a concentration of lxl O6 / mL, c ) when 90% confluent , grown separately in 24 , 48 and 72 hour periods and the media in each time period are collected, d) using the collected media samples for obtaining the final product by mixing them with water-based cream, gel , spray and serum bases in 5 , 10 and 20% volumes in lyophili zed or crude formulations of cream, gel , spray and serum formulations .

[0023] In an embodiment of the invention, media are firstly derived from mammalian macrophage cells in the method for producing the product of the invention, which media are formed using an RPMI medium (RPMI 1640 ) containing 10% Fetal Bovine Serum ( FBS ) and 1 % Penicillin-streptomycin .

[0024] In an embodiment of the invention, mammalian cells in these media are grown until 90% confluent at a concentration of lxl O6 / mL .

[0025] In an embodiment of the invention, the media are collected in slices of 24 , 48 , and 72 hours when 90% confluent .

[0026] In one embodiment of the invention, the collected media samples are used for obtaining the final product by mixing with waterbased creams , gels , sprays and serum bases in 5 , 10 and 20% by volume in the formulations of creams , gels , sprays and serum bases in their lyophili zed or raw form .

[0027] In one embodiment of the invention, the final product is provided in the form of a water-based cream, gel , spray, or serum base .

[0028] In an embodiment of the invention, the crude or lyophili zed media are combined with biocompatible solutions to produce a dermocosmetic product to obtain the final product .

[0029] The product obtained by the method of the invention includes , inter alia, growth signals produced by macrophages taken from mammalian cells , anti-apoptotic signals and signals to support the survival of cells . Growth signals produced by macrophages from mammalian cells , anti-apoptotic signals growth factors , signals to support cell survival , and molecules enable other cells in the environment ( as detected in epithelium and fibroblast cells ) to grow, proli ferate , and repair . The mixture obtained from the cells is used as a biotechnological product in cream, gel , spray and serum formulations for wound repair, antiaging and beauty purposes . The product obtained by the method of the invention comprises each of the following in concentrations ranging from 100 pg / mL to lOOOpg / mL at natural production rates: platelet-derived growth factor (PDGF) , macrophage-derived growth factor (MDGF) , bone morphogenetic proteins (BMP 2, 6, 7) family members, transforming growth factor p family members, endothelial growth factor (EGF) family members, insulin-like growth factor (IGF) family members, members of the vascular endothelial growth factor (VEGF) family, fibroblast growth factor (EGF) family members .

[0030] The product obtained by the method according to an embodiment of the invention does not contain any chemical agents or preservatives other than biocompatible solutions in cream, spray, gel and serum bases. Said biocompatible solutions preferably include, but are not limited to, Hank's balanced salt solution (HBSS) , phosphate buffered salt solution (PBS) .

[0031] The product obtained by the method according to an embodiment of the invention does not contain materials with low efficiency such as collagen or urea. Thus, the toxic or allergic effects of the product are reduced.

[0032] Since the mammalian macrophage cell-based biotechnological products (or active ingredients) of the invention are biocompatible and have high biological functions, their effectiveness is also high.

[0033] The method of the invention provides a product with a long shelf life is obtained without using a protective product. In addition, the product obtained by the method according to the invention is produced in the laboratory through mammalian cells and therefore, unlike the methods that cause a decrease in the population of plants or living things, the method of the invention offers an environmentally friendly production. In the present disclosure of the present invention, the production method of the invention is illustrated by nonlimiting examples , which are provided only for a better understanding of the subj ect matter, and it is clear that modi fications can be made by those skilled in the art without departing from the scope of the claims .

Claims

AMENDED CLAIMS received by the International Bureau on 29 April 2024 (29.04.2024)1. A method for manufacturing a biotechnological product for dermocosmetic purposes, comprising a. preparation of a conditioned media to be obtained from mammalian macrophage cell lines, b. growing mammalian macrophage cells until confluency and producing factors at certain rates, c. collection of the confluent media at specific time points while the mammalian macrophage cells are confluent; and d. obtaining a final product by mixing the collected media with a material containing at least one of a group of cream, gel, spray, serum base in crude and / or lyophilized form.

2. The method of claim 1, wherein the media in the step a is formed by using RPMI medium containing 10% fetal bovine serum and 1% penicilin-streptomycin .

3. The method of claim 1, wherein the mammalian macrophage cells are grown at a concentration of lxlO6 / mL in step b.

4. The method of claim 1 or 3, wherein the mammalian macrophage cells are grown until 90% confluency in step b and the media is collected in 24, 48 and / or 72 hours in step c.

5. The method of claim 1, wherein the collected media are mixed in 5%, 10% and / or 20% by volume with the material containing at least one of the group of cream, gel, spray, serum base in step d.

6. A biotechnological product obtained by a method according to any one of the preceding claims.

7. The biotechnological product of claim 6, wherein it comprises10AMENDED SHEET (ARTICLE 19)each of following in concentrations ranging from 100 pg / mL to lOOOpg / mL at natural production rates: platelet-derived growth factor (PDGF) , macrophage-derived growth factor (MDGF) , bone morphogenetic proteins (BMP 2, 6, 7) family members, transforming growth factor p family members, endothelial growth factor (EGF) family members, insulin-like growth factor (IGF) family members, members of the vascular endothelial growth factor (VEGF) family, fibroblast growth factor (EGF) family members.11AMENDED SHEET (ARTICLE 19)